Methods of producing 6-carbon chemicals using 2,6-diaminopimelate as precursor to 2-aminopimelate

ABSTRACT

This document describes biochemical pathways for producing 2-aminopimelate from 2,6-diaminopimelate, and methods for converting 2-aminopimelate to one or more of adipic acid, adipate semialdehyde, caprolactam, 6-aminohexanoic acid, 6-hexanoic acid, hexamethylenediamine, or 1,6-hexanediol by decarboxylating 2-aminopimelate into a six carbon chain aliphatic backbone and enzymatically forming one or two terminal functional groups, comprised of carboxyl, amine or hydroxyl group, in the backbone.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.14/714,164, filed May 15, 2015, which claims priority to U.S.Application Ser. No. 61/993,532, filed on May 15, 2014, the disclosuresof which are incorporated by reference in their entireties.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

An official copy of the sequence listing is submitted electronically viaEFS-Web as an ASCII formatted sequence listing with a file named“12444_0286-01000_SL.txt”, created on Jul. 6, 2017. Said ASCII copy is153,047 bytes in size. The sequence listing contained in this ASCIIformatted document is part of the specification and is hereinincorporated by reference in its entirety.

TECHNICAL FIELD

Disclosed herein are methods for biosynthesizing 2-aminopimelate in arecombinant host from 2,6-diaminopimelate using one or more of apolypeptide having 2-hydroxyacyl-CoA dehydratase activity, a polypeptidehaving mutase activity, a polypeptide having ammonia lyase activity, anda polypeptide having enoale reductase activity. The biosynthesized2-aminopimelate can be enzymatically converted to a product selectedfrom the group consisting of adipic acid, adipate semialdehyde,6-aminohexanoic acid, 6-hydroxyhexanoic acid, caprolactam,hexamethylenediamine, and 1,6-hexanediol using, for example, one or moreof a polypeptide having α-oxoacid decarboxylase activity classifiedunder EC 4,1.1.-, a polypeptide having α-aminoacid decarboxylaseactivity classified under EC 4.1.1.-, a polypeptide having synthaseactivity, and a polypeptide having the activity of a dehydrogenasecomplex; and one or more optional polypeptides having an activity suchas aldehyde dehydrogenase activity, alcohol dehydrogenase activity,CoA-transferase activity, carboxylate reductase activity,α-aminotransferase activity, thioesterase activity, hydrolase activity,ω-transaminase activity, N-acetyltransferase activity, or deacylaseactivity, and combinations thereof.

BACKGROUND

Nylons are polyamides which are sometimes synthesized by thecondensation polymerisation of a diamine with a dicarboxylic acid.Similarly, nylons may be produced by the condensation polymerisation oflactams. A ubiquitous nylon is nylon 6,6, which is produced by reactionof hexamethylenediamine (HMD) and adipic acid. Nylon 6 is produced by aring opening polymerisation of caprolactam. Therefore, adipic acid,hexamethylenediamine, and caprolactam are important intermediates in theproduction of nylons (Anton & Baird, Polyamides Fibers, Encyclopedia ofPolymer Science and Technology, 2001).

Industrially, adipic acid and caprolactam are produced via air oxidationof cyclohexane. The air oxidation of cyclohexane produces, in a seriesof steps, a mixture of cyclohexanone (K) and cyclohexanal (A),designated as KA oil, Nitric acid oxidation of KA oil produces adipicacid (Musser, Adipic acid, Ullmann's Encyclopedia of IndustrialChemistry, 2000). Caprolactam is produced from cyclohexanone via itsoxime and subsequent acid rearrangement (Fuchs, Kieczka and Moran,Caprolactam, Ullmann's Encyclopedia of Industrial Chemistry, 2000).

Industrially, hexamethylenediamine (HMI)) is produced by hydrocyanationof C6 Building Block to adiponitrile, followed by hydrogenation to HMD(Herzog and Smiley, Hexamethylenediamine, Ullmann's Encyclopedia ofIndustrial Chemistry, 2012).

Given a reliance on petrochemical feedstocks; biotechnology offers analternative approach via biocatalysis. Biocatalysis is the use ofbiological catalysts, such as enzymes, to perform biochemicaltransformations of organic compounds.

Both bioderived feedstocks and petrochemical feedstocks are via startingmaterials for the biocatalysis processes.

Accordingly, against this background, it is clear that there is a needfor sustainable methods for producing adipic acid, caprolactam,6-aminohexanoic acid, hexamethylenediamine and 1,6-hexanediol (hereafter“C6 building blocks”) wherein the methods are biocatalyst-based (Jang etal., Biotechnology & Bioengineering, 2012, 109(10), 2437-2459).

However, no wild-type prokaryote or eukaryote naturally overproduces orexcretes C6 building blocks to the extracellular environment.Nevertheless, the metabolism of adipic acid and caprolactam has beenreported (Ramsay et al., Appl. Environ. Microbiol., 1986, 52(1),152-156; Kulkarni and Kanekar, Current Microbiology, 1998, 37, 191 194),

The dicarboxylic acid, adipic acid, is converted efficiently as a carbonsource by a number of bacteria and yeasts via β-oxidation into centralmetabolites. β-oxidation of o adipate to 3-oxoadipate faciliates furthercatabolism via, for example, the ortho-cleavage pathway associated witharomatic substrate degradation. The catabolism of 3-oxoadipyl-CoA toacetyl-CoA and succinyl-CoA by several bacteria and fungi has beencharacterised comprehensively (Harwood and Parales, Annual Review ofMicrobiology, 1996, 50, 553-590). Both adipate and 6-aminohexanoic acidare intermediates in the catabolism of caprolactam, finally degraded via3-oxoadipyl-CoA to central metabolites.

Potential metabolic pathways have been suggested for producing adipicacid from biomass-sugar: (1) biochemically from glucose to cis,cismuconic acid via the ortho-cleavage aromatic degradation pathway,followed by chemical catalysis to adipic acid; (2) a reversible adipicacid degradation pathway via the condensation of succinyl-CoA andacetyl-CoA and (3) combining β-oxidation, fatty acid synthase, andω-oxidation. However, no information using these strategies has beenreported (Jang et al., Biotechnology & Bioengineering, 2012, 109(10),2437-2459).

An optimality principle states that microorganisms regulate theirbiochemical networks to support maximum biomass growth. Beyond the needfor expressing heterologous pathways in a host organism, directingcarbon flux towards C6 building blocks that serve as carbon sourcesrather than as biomass growth constituents, contradicts the optimalityprinciple. For example, transferring the 1-butanol pathway fromClostridium species into other production strains has often fallen shortby an order of magnitude compared to the production performance ofnative producers (Shen et al., Appl. Environ. Microbiol., 2011, 77(9),2905-2915).

The efficient synthesis of a six or seven carbon aliphatic backbone ascentral precursor is a key consideration in synthesizing C6 buildingblocks prior to forming terminal functional groups, such as carboxyl,amine or hydroxyl groups, on the C6 aliphatic backbone.

SUMMARY

This document is based, at least in part, on the discovery that it ispossible to construct biochemical pathways for producing a seven carbonchain aliphatic backbone as a central precursor, which can bedecarboxylated to a six carbon aliphatic backbone in which one or twofunctional groups, i.e., carboxyl, amine or hydroxyl, can be formed,leading to the synthesis of adipic acid, adipate semialdehyde,6-aminohexanoic acid, 6-hydroxyhexanoate, hexamethylenediamine,caprolactam, or 1,6-hexanediol (hereafter “C6 building blocks). Adipicacid and adipate, 6-hydroxyhexanoic acid and 6-hydroxyhexanoate, and6-aminohexanoic acid and 6-aminohexanoate are used interchangeablyherein to refer to the compound in any of its neutral or ionized forms,including any salt forms thereof. It is understood by those skilled inthe art that the specific form will depend on pH. These pathways,metabolic engineering, and cultivation strategies described herein usemeso-2,6 diaminopimelate as a central metabolite, which can heenzymatically converted to (S) 2-aminopimelate or (R) 2-aminopimelate.

In the face of an optimality principle, surprisingly it has beendiscovered that appropriate non-natural pathways, feedstocks, hostmicroorganisms, attenuation strategies to the host's biochemical networkand cultivation strategies may be combined to efficiently produce one ormore C6 building blocks.

In one aspect, this document features a method of biosynthesizing2-aminopimelate in a recombinant host. The method includes enzymaticallyconverting 2,6-diaminopimelate to 2-aminopimelate in the host using atleast one polypeptide having an activity selected from the groupconsisting of 2-hydroxyacyl-CoA dehydratase activity, mutase activity,ammonia lyase activity, and enoate reductase activity. In someembodiments, the method can include enzymatically converting2,6-diaminopimelate to (S) 2-aminopimelate. In some embodiments, themethod can include enzymatically converting 2,6-diaminopimelate to (R)2-aminopimelate. The method can include using a polypeptide having2-hydroxyacyl-CoA dehydratase activity and a polypeptide having enoatereductase activity to enzymatically convert 2,6-diaminopimelate to2-aminopimelate. The polypeptide having 2-hydroxyacyl-CoA dehydrataseactivity can have at least 70%, at least 80%, or at least 90% sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 25 or SEQ IDNO: 28. The poly peptide having enoate reductase activity can have atleast 70%, at least 80%, or at least 90% sequence identity to the aminoacid sequence set forth in any one of SEQ ID NOs: 16-22. The method cano include using a polypeptide having mutase activity, a polypeptidehaving ammonia lyase activity, a said polypeptide having enoatereductase activity to enzymatically convert 2,6-diaminopimelate to2-aminopimelate. The polypeptide having ammonia lyase activity can haveat least 70%, at least 80%, or at least 90% sequence identity to theamino acid sequence set forth in SEQ ID NO: 23. The polypeptide havingmutase activity has at least 70%, at least 80%, or at least 90% sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 26.

The method disclosed can further include using at least one polypeptidehaving an activity selected from the group consisting of diaminopimelatedehydrogenase activity, 2-hydroxycarboxylate dehydrogenase activity,CoA-transferase activity, 2-hydroxyacid dehydratase activity, andcarbaxylate reductase activity to enzymatically convert2,6-diaminopimelate to 2-aminopimelate. The methods disclosed canfurther include using using at least one polypeptide having an activityselected from the group consisting of CoA ligase activity,CoA-transferase activity, carboxylate reductase activity, and aldehydedehydrogenase activity to enzymatically convert 2,6-diaminopimelate to2-aminopimelate.

In some embodiments, the central precursor comprises a C7 aliphaticbackbone such as S)-2-aminopimelate or (R)-2-aminopimelate, forenzymatic conversion to one or more C6 building blocks. Such C7aliphatic backbones can be formed from a lysine biosynthesis precursorsuch as meso-2,6 diaminopimelate. See FIG. 1 and FIG. 2.

In some embodiments, a terminal carboxyl group can be enzymaticallyformed using a thioesterase, a CoA-transferase or CoA-ligase, or analdehyde dehydrogenase. See FIG. 3.

In some embodiments, a terminal amine group can be enzymatically formedusing an (R) alpha-aminodecarboxylase (classified, for example, under EC4.1.1,—such as EC 4.1.1.20), (S) alpha-aminodecarboxylase (classified,for example, under EC 4.1.1.—such as EC 4.1.1.15, EC 4.1.1.17 or EC4.1.1.18) or a transaminase (classified, for example, under EC 2.6.1.-).See FIG. 4, FIG. 5, FIG. 6, and FIG. 7.

In some embodiments, a terminal hydroxyl group can be enzymaticallyformed o using a NADPH-specific or NADH-specific alcohol dehydrogenase.See FIG. 8.

In some embodiments, the principal carbon source fed to the fermentationderived from a biological feedstock or a non-biological feedstock.

In some embodiments, the biological feedstock can be or can derive from,monosaccharides, disaccharides, lignocellulose, hemicellulose,cellulose, lignin, levulinic acid and formic acid, triglycerides,glycerol, fatty acids, agricultural waste, condensed distillers'solubles, or municipal waste.

In some embodiments, the non-biological feedstock can be or can derivefrom natural gas, syngas, CO₂H₂, methanol, ethanol, benzoate,non-volatile residue (NVR) or a caustic wash waste stream fromcyclohexane oxidation processes, or terephthalic acid/isophthalic acidmixture waste streams.

In some embodiments, the host microorganism is a prokaryote. Forexample, the prokaryote can be from the bacterial genus Escherichia suchas Escherichia coli; from the bacterial genus Clostridia such asClostridium ljungdahlii, Clostridium autoethanogenum or Clostridiumkluyveri; from the bacterial genus Corynebacteria such asCorynebacterium glutamicum, from the bacterial genus Cupriavidus such asCupriavidus necator or Cupriavidus metallidurans; from the bacterialgenus Pseudomonas such as Pseudomonas fluorescens, Pseudomonas putida orPseudomonas oleavorans; from the bacterial genus Delftia such as Delftiaacidovorans; from the bacterial genus Bacillus such as Bacillussubtillis; from the bacterial genus Lactobacillus such as Lactobacillusdelbrueckii, or from the bacterial genus Lactococcus such as Lactococcuslactis. Such prokaryotes also can be a source of genes to constructrecombinant host cells described herein that are capable of producingone or more C6 building blocks.

In some embodiments, the host microorganism is a eukaryote (e.g., afungus such as a yeast). For example, the eukaryote can be from thefungus genus Aspergillus such as Aspergillus niger; from the yeast genusSaccharomyces such as Saccharomyces cerevisiae; from the yeast genusPichia such as Pichia pastoris; from the yeast genus Yarrowia such asYarrowia lipolytica; from the yeast genus Issatchenkia such asIssathenkia orientalis; from the yeast genus Debaryomyces such asDebaryomyces hansenii; from the yeast genus Arxula such as Arxulaadenoinivorans; or from the yeast genus Kluyveromyces such asKluyveromyces lactis. Such eukaryotes also can be a source of genes toconstruct recombinant host cells described herein that are capable ofproducing one or more C6 building blocks.

The reactions of the pathways described herein can be performed in oneor more cell (e.g., host cell) strains (a) naturally expressing one ormore relevant enzymes, (b) genetically engineered to express one or morerelevant enzymes, or (c) naturally expressing one or more relevantenzymes and genetically engineered to express one or more relevantenzymes. Alternatively, relevant enzymes can be extracted from any ofthe above types of host cells and used in a purified or semi-purifiedform. Extracted enzymes can optionally be immobilized to a solidsubstrate such as the floors and/or walls of appropriate reactionvessels. Moreover, such extracts include lysates (e.g. cell lysates)that can be used as sources of relevant enzymes. In the methods providedby the document, all the steps can be performed in cells (e.g., hostcells), all the steps can be performed using extracted enzymes, or someof the steps can be performed in cells and others can be performed usingextracted enzymes.

Many of the enzymes described herein catalyze reversible reactions, andthe reaction of interest may be the reverse of the described reaction.The schematic pathways shown in FIGS. 1-8 illustrate the reaction ofinterest for each of the intermediates.

In some embodiments, the host microorganism's tolerance to highconcentrations of a C6 building block is improved through continuouscultivation in a selective environment.

In some embodiments, the host microorganism's biochemical network isattenuated or augmented to (1) ensure the intracellular availability ofoxaloacetate, (2) create an NADPH imbalance that may only be balancedvia the formation of one or more C6 building blocks, (3) preventdegradation of central metabolites or central precursors leading to andincluding C6 building blocks and (4) ensure efficient efflux from thecell.

In some embodiments, the cultivation strategy entails either achievingan aerobic or micro-aerobic cultivation condition.

In some embodiments, the cultivation strategy entails nutrientlimitation either via nitrogen, phosphate or oxygen limitation.

In some embodiments, the cultivation strategy entails preventing theincorporation of fatty acids into lipid bodies or other carbon storageunits.

In some embodiments, one or more C6 building blocks are produced by asingle type of microorganism, e.g., a recombinant host containing one ormore exogenous nucleic acids, using, for example, a fermentationstrategy.

In some aspects, the methods disclosed further comprising enzymaticallyconverting 2-aminopimelate to a product selected from the groupconsisting of adipic acid, adipate semialdehyde, 6-aminohexanoic acid,6-hydroxyhexanoic acid, caprolactam, hexamethylenediamine, and1,6-hexanediol. The method includes enzymatically converting2-aminopimelate to one or more of said products using (i) at least onepolypeptide having an activity selected from the group consisting ofα-oxoacid decarboxylase activity classified under EC 4.1.1.-,α-aminoacid decarboxylase activity classified under EC 4.1.1.-, synthaseactivity, and activity of a dehydrogenase complex; and (ii) one or moreoptional polypeptides having an activity selected from the groupconsisting of aldehyde dehydrogenase activity, alcohol dehydrogenaseactivity, CoA-transferase activity, carboxylate reductase activity,α-aminotransferase activity, thioesterase activity, hydrolase activity,ω-transaminase activity, N-acetyltransferase activity, and deacylaseactivity. The polypeptide having α-oxoacid decarboxylase activity can beclassified under EC 4.1.1.43, EC 4.1.1.71, EC 4.1.1.72 or EC 4.1.1.74.The polypeptide having α-aminoacid decarboxylase activity can beclassified under EC 4.1.1.15, EC 4.1.1.17, EC 4.1.1.18, EC 4.1.1.19. Thepolypeptide having synthase activity is classified under EC 2.2.1.6, orthe polypeptide having the activity of a dehydrogenase complex comprisesactivities can be classified under EC 1.2.4.2, EC 1.8.1.4 and EC2.3.1.61.

For example, the methods disclosed herein further can includedenzymatically converting 2-aminopimelate to adipic acid using at leastone polypeptide having an activity selected from the group consisting ofα-aminotransferase activity, 2-exoacid decarboxylase activity, synthaseactivity, dehydrogenase complex activity, thioesterase activity,CoA-transferase activity, CoA-ligase activity, and aldehydedehydrogenase activity.

For example, the methods disclosed herein further can includedenzymatically converting 2-aminopimelate to adipate semialdehyde usingat least one polypeptide having an activity selected from the groupconsisting of α-aminotransferase activity, 2-oxoacid decarboxylaseactivity, and synthase activity.

For example, the methods disclosed herein further can includedenzymatically converting 2-aminopimelate to 6-aminohexanoic acid using apolypeptide having α-aminoacid decarboxylase activity.

For example, the methods disclosed herein further can includedenzymatically converting adipate semialdehyde to 6-aminohexanoic fromusing a ω-transaminase. The methods can further include biosynthesizingcaprolactam from 6-aminohexanoic acid using a polypeptide having theactivity of a hydrolase.

For example, the methods disclosed herein further can includedenzymatically converting 6-aminohexanoic acid to hexamethylenediaminefrom using at least one polypeptide having an activity selected from thegroup consisting of carboxylate reductase activity, N-acetyltransferaseactivity, ω-transaminase activity, and deacylase activity.

For example, the method further can include enzymatically convertingadipate semialdehyde to hexamethylenediamine using at least onepolypeptide having an selected from the group consisting of carboxylatereductase activity and ω-transaminase activity.

For example, the methods disclosed herein further can includedenzymatically converting 2-aminopimelate to 6-hydroxyhexanoic acid usingat least one polypeptide having an activity selected from the groupconsisting of α-aminotransferase activity, 2-oxoacid decarboxylaseactivity, synthase activity, and alcohol dehydrogenase activity.

For example, the methods disclosed herein further can includedenzymatically converting 6-hydroxyhexanoic acid to hexamethylenediamineusing at least one o polypeptide having an activity selected from thegroup consisting of carboxylate reductase activity, ω-transaminaseactivity, and alcohol dehydrogenase activity.

For example, the methods disclosed herein further can includedenzymatically converting 6-hydroxyhexanoic acid to 1,6-hexanediol usinga polypeptide having carboxylate reductase activity and a polypeptidehaving alcohol dehydrogenase activity.

The polypeptide having 2-oxoacid decarboxylase activity can have atleast 70% sequence identity to the amino acid sequence set forth in SEQID NO:34, the polypeptide having α-aminoacid decarboxylase activity canhave at least 70% sequence identity to the amino acid sequence set forthin any one of SEQ ID NOs: 29-34.

The polypeptide having carboxylate reductase activity can have at least70% sequence identity to the amino acid sequence set forth in any one ofSEQ ID NOs: 3-7.

The polypeptide having ω-transaminase activity can have at least 70%sequence identity to the amino acid sequence set forth in any one of SEQID NOs: 8-13.

The polypeptide having thioesterase activity can have at least 70%sequence identity to the amino acid sequence set forth in SEQ ID NO: 1or SEQ ID NO: 2.

In some embodiments, the host comprises one or more of the following:the intracellular concentration of oxaloacetate for biosynthesis of a C6building block is increased in the host by overexpressing recombinantgenes forming oxaloacetate; wherein an imbalance in NADPH is generatedthat can be balanced via the formation of a C6 building block; whereinan exogenous lysine biosynthesis pathway synthesizing lysine from2-oxoglutarate via 2-oxoadipate is introduced in a host using the meso2,6 diaminopimelate pathway for lysine synthesis; wherein an exogenouslysine biosynthesis pathway synthesizing lysine from oxaloacetate tomeso 2,6 diaminopimelate is introduced in a host using the 2-oxoadipatepathway for lysine synthesis; wherein endogenous degradation pathways ofcentral metabolites and central precursors leading to and including C6building blocks are attenuated in the host; or wherein the efflux of aC6 building block across the cell membrane to the extracellular media isenhanced or amplified by genetically engineering structuralmodifications to the cell membrane or increasing any associatedtransporter activity for a C6 building block.

This document also features a recombinant host that includes at leastone o exogenous nucleic acid encoding at least one polypeptide having anactivity selected from the group consisting of 2-hydroxyacyl-CoAdehydratase activity, mutase activity, ammonia lyase activity, andenoate reductase activity, said host producing 2-aminopimelate from2,6-diaminopimelate. For example, the recombinant host can include apolypeptide having exogenous 2-hydroxyacyl-CoA dehydratase activity anda polypeptide having enoate reductase activity. For example, therecombinant host can include a polypeptide having mutase activity, apolypeptide having ammonia lyase activity, and a polypeptide havingenoate reductase activity. The polypeptide having enoate reductaseactivity can have at least 70% sequence identity to the amino acidsequence set forth in any one of SEQ ID NOs: 16-22. The polypeptidehaving 2-hydroxyacyl-CoA dehydratase activity can have at least 70%sequence identity to the amino acid sequence set forth in SEQ ID NO: 25or SEQ ID NO: 28. The polypeptide having mutase activity can have atleast 70% sequence identity to the amino acid sequence set forth in SEQID NO: 26. The polypeptide having ammonia lyase activity can have atleast 70% sequence identity to the amino acid sequence set forth in SEQID NO: 23.

The host can further include at least one or more exogenous polypeptideshaving an activity selected from the group consisting of a)diaminopimelate dehydrogenase activity, 2-hydroxycarboxylatedehydrogenase activity, CoA-transferase activity, 2-hydroxyaciddehydratase activity, and carboxylate reductase activity; or b) CoAligase activity, CoA-transferase activity, carboxylate reductaseactivity, and aldehyde dehydrogenase activity.

The host can further include at least one or more exogenous polypeptideshaving an activity selected from the group consisting of α-oxoaciddecarboxylase activity classified under EC 4.1.1.-, α-aminoaciddecarboxylase activity classified under EC 4.1,1.-, synthase activity,and activity of a dehydrogenase complex.

The host can further include at least one or more exogenous polypeptideshaving an activity selected from the group consisting of aldehydedehydrogenase activity, alcohol dehydrogenase activity, CoA-transferaseactivity, carboxylate reductase activity, α-aminotransferase activity,thioesterase activity, hydrolase activity, ω-transaminase activity,N-acetyltransferase activity, and deacylase activity, the host producinga product selected from the group consisting of adipic acid, adipatesemialdehyde, 6-aminohexanoic acid, 6-hydroxyhexanoic acid, caprolactam,hexamethylenediamine, and 1,6-hexanediol.

The host can further include at least one or more exogenous polypeptideshaving an an activity selected from the group consisting ofα-aminotransferase activity, 2-oxoacid decarboxylase activity, activityof a dehydrogenase complex, thioesterase activity, CoA-transferaseactivity, CoA-ligase activity, and aldehyde dehydrogenase activity, thehost producing adipic acid.

The host can further include at least one or more exogenous polypeptideshaving an activity selected from the group consisting ofα-aminotransferase activity, 2-oxoacid decarboxylase activity, synthaseactivity, the host producing adipate semialdehyde.

The host can further include at least one or more exogenous polypeptideshaving an α-aminoacid decarboxylase activity, the host producing6-aminohexanoic acid.

A recombinant host producing 6-aminohexanoic acid can include anexogenous polypeptide having ω-transaminase activity. A recombinant hostproducing 6-aminohexanoic acid further can include an exogenouspolypeptide having hydrolase activity, the host producing caprolactam.The host can further include one or more of an exogenous polypeptidehaving carboxylate reductase activity, N-acetyltransferase activity,ω-transaminase activity, or deacylase activity, the host producinghexamethylenediamine.

The host cell can further include at least one exogenous polypeptidehaving carboxylate reductase activity and/or at least one exogenouspolypeptide having ω-transaminase activity, the host producinghexamethylenediamine.

The host cell can further include at at least one exogenous polypeptidehaving an activity selected from the group consisting ofα-aminotransferase activity, α-oxoacid decarboxylase activity, alcoholdehydrogenase activity or synthase activity, the host producing6-hydroxyhexanoic acid.

The host cell can further include at least one exogenous polypeptidehaving an activity selected from the group consisting of carboxy/atereductase activity, ω-transaminase activity, and alcohol dehydrogenaseactivity, the host producing hexamethylenediamine.

The host cell can further include at an exogenous polypeptide havingcarboxylase reductase activity and/or an exogenous polypeptide havingalcohol dehydrogenase activity, the host producing 1,6-hexanediol.

In one aspect, this document features a method for producing abioderived 6-carbon compound. The method for producing a bioderived6-carbon compound can include culturing or growing a recombinant host asdescribed herein under conditions and for a sufficient period of time toproduce the bioderived 6-carbon compound, wherein, optionally, thebioderived 6-carbon compound is selected from the group consisting ofadipic acid, adipate semialdehyde, 6-aminohexanoic acid,6-hydroxyhexanoic acid, caprolactam, hexamethylenediamine,1,6-hexanediol, and combinations thereof.

In one aspect, this document features composition comprising abioderived 6-carbon compound as described herein and a compound otherthan the bioderived 6-carbon compound, wherein the bioderived 6-carboncompound is selected from the group consisting of adipic acid, adipatesemialdehyde, 6-aminohexanoic acid, 6-hydroxyhexanoic acid, caprolactam,hexamethylenediamine, 1,6-hexanediol, and combinations thereof. Forexample, the bioderived 6-carbon compound is a cellular portion of ahost cell or an organism.

This document also features a biobased polymer comprising the bioderivedadipic acid, adipate semialdehyde, 6-aminohexanoic acid,6-hydroxyhexanoic add, caprolactam, hexamethylenediamine,1,6-hexanediol, and combinations thereof.

This document also features a biobased resin comprising the bioderivedadipic acid, adipate semi aldehyde, 6-aminohexanoic acid.6-hydroxyhexanoic acid, caprolactam, hexamethylenediamine,1,6-hexanediol, and combinations thereof, as well as a molded productobtained by molding a biobased resin.

In another aspect, this document features a process for producing abiobased polymer that includes chemically reacting the bioderived adipicacid, adipate o semialdehyde, 6-aminohexanoic acid, 6-hydroxyhexanoicacid, caprolactam, hexamethylenediamine, 1,6-hexanediol, with itself oranother compound in a polymer producing reaction.

In another aspect, this document features a process for producing abiobased resin that includes chemically reacting the bioderived adipicacid, adipate semialdehyde, 6-aminohexanoic acid, 6-hydroxyhexanoicacid, caprolactam, hexamethylenediamine, 1,6-hexanediol, with itself oranother compound in a resin producing reaction.

Any of the recombinant hosts described herein further can includeattenuation of one or more of the following enzymes: apolyhydroxyalkanoate synthase, an acetyl-CoA thioesterase, aphosphotransacetylase forming acetate, an acetate kinase, a lactatedehydrogenase, a menaquinol-fumarate oxidoreductase, an alcoholdehydrogenase forming ethanol, a those phosphate isomerase, a pyruvatedecarboxylase, a glucose-6-phosphate isomerase, NADH-consumingtranshydrogenase, an NADH-specific glutamate dehydrogenase, aNADH/NADPH-utilizing glutamate dehydrogenase, a pimeloyl-CoAdehydrogenase; an acyl-CoA dehydrogenase accepting C6 building blocksand central precursors as substrates; a butyl-CoA dehydrogenase; or anadipyl-CoA synthetase.

Any of the recombinant hosts described herein further can overexpressone or more genes encoding: 2-hydroxyacyl-CoA dehydratase; a mutase; aCoA-ligase; an ammonia lyase; an acetyl-CoA synthetase; an enoatereductase; a 6-phosphogluconate dehydrogenase; a transketolase; apuridine nucleotide transhydrogenase; a glyceraldehyde-3P-dehydrogenase;a malic enzyme; a glucose-6-phosphate dehydrogenase; a glucosedehydrogenase; a fructose 1,6 diphosphatase; a L-alanine dehydrogenase;a L-glutamate dehydrogenase; a formate dehydrogenase; a L-glutaminesynthetase; a diamine transporter; a dicarboxylate transporter;diaminopimelate dehydrogenase; 2-hydroxycarboxylate dehydrogenase,2-hydroxyacid dehydratase, carboxylate reductase and/or a multidrugtransporter.

Also, described herein is a biochemical network comprising adehydrogenase, a CoA-transferase, a dehydratase, a reductase, a mutase,a CoA-ligase, an ammonia lyase, or a thioesterase andmeso-2,6-diaminopimelate, wherein the dehydrogenase, theCoA-transferase, the dehydratase, the reductase, the mutase, theCoA-ligase, the ammonia lyase, or the thioesterase enzymaticallyconverts the meso-2,6-diaminopimelate to 2-aminopimelate. Thebiochemical network can further include an α-aminotransferase, whereinthe aminotransferase enzymatically converts 2-aminopimelate to2-oxo-pimelate. The biochemical network can further include adecarboxylase, a synthase, or a dehydrogenase complex, wherein thedecarboxylase, the synthase, or the dehydrogenase complex enzymaticallyconverts 2-oxo-pimelate to adipyl-CoA or adipate semialdehyde. Thebiochemical network can further include a dehydrogenase, a CoAtransferase, a CoA ligase or a thioesterase, wherein the dehydrogenase,the CoA transase, the CoA ligase, or the thioesterase enzymaticallyconvert adipyl-CoA or adipate semialdehyde to adipic acid.

Also, described herein a biochemical network comprising dehydrogenase, aCoA-transferase, a dehydratase, a reductase, a mutase, a CoA-ligase, anammonia lyase, or a thioesterase and meso-2,6-diaminopimelate, whereinthe dehydrogenase, the CoA-transferase, the dehydratase, the reductase,the mutase, the CoA-ligase, the ammonia lyase, or the thioesteraseenzymatically converts the meso-2,6-diaminopimelate to 2-aminopimelate.The biochemical network can further include a decarboxylase; wherein thedecarboxylase enzymatically converts 2-aminopimelate to 6-aminohexanoicacid. The biochemical network can further include a hydrolase, areductase (e.g., a carboxylate reductase), a transaminase, anN-acetyltransferase, or a deacylase, wherein the hydrolase, thereductase, the transaminase, the N-acetyltransferase, or the deacetylaseenzymatically convert 6-aminohexanoic acid into at least one ofcaprolactam or hexamethylenediamine.

Also, described herein is a biochemical network comprising adehydrogenase, a CoA-transferase, a dehydratase, a reductase, a mutase,a CoA-ligase, an ammonia lyase, or a thioesterase andmeso-2,6-diaminopimelate, wherein the dehydrogenase, theCoA-transferase, the dehydratase, the reductase, the mutase, theCoA-ligase, the ammonia lyase, or the thioesterase enzymaticallyconverts the meso-2,6-diaminopimelate to 2-aminopimelate. Thebiochemical network can further include an aminotransferase, a osynthase, a decarboxylase, or a dehydrogenase wherein theaminotransferase, the synthase, the decarboxylase, or the dehydrogenaseenzymatically converts 2-aminopimelate to 6-hydroxyhexanoic acid. Thebiochemical network can further include a reductase (e.g., a carboxylatereductase), a transaminase, or an alcohol dehydrogenase, wherein thereductase, the transaminase, or the alcohol dehydrogenase enzymaticallyconvert 6-hydroxyhexanoic acid into at least one of hexamethylenediamineand 1,6-hexanediol.

Also, described herein is a means for obtaining 2-aminopimelate using atleast one of a dehydrogenase, a CoA-transferase, a dehydratase, areductase, a mutase, a CoA-ligase, an ammonia lyase, or a thioesterase.The means can further include means for converting 2-aminopimelate to atleast one of adipic acid, 6-aminohexanoic acid, caprolactam,hexamethylenediamine, 6-hydroxyhexanoic acid, and 1,6-hexanediol. Themeans can include a decarboxylase, a synthase, a dehydrogenase complex,a dehydrogenase, a CoA-transferase, a dehydratase, a reductase, amutase, a CoA-ligase, a lyase, a thioesterase, an aminotransferase, ahydrolase, a transaminase, or an N-acetyltransferase.

Also described herein is (i) step for obtaining 2-aminopimelate using adehydrogenase, a CoA-transferase, a dehydratase, a reductase, a mutase,a CoA-ligase, an ammonia lyase, or a thioesterase (ii) a step forobtaining adipic acid using a decarboxylase, a synthase, or adehydrogenase complex; (iii) a step for obtaining 6-aminohexanoic acidusing a decarboxylase; and (iv) a step for obtaining 6-hydroxyhexanoicacid using a at least one of a aminotransferase, a synthase, adecarboxylase, or a dehydrogenase.

In another aspect, this document features a composition comprising2-aminopimelate and decarboxylase, a synthase, or a dehydrogenasecomplex. The composition can be cellular. The composition can furtherinclude a dehydrogenase, a CoA-transferase, a CoA-dehydratase, adehydratase, a reductase, a mutase, a CoA-ligase, a lyase, athioesterase, an aminotransferase, a hydrolase, a transaminase, or anN-acetyltransferase and at least one of adipic acid, 6-aminohexanoicacid, caprolactam, hexamethylenediamine, 6-hydroxyhexanoic acid, and1,6-hexanediol. The composition can be cellular.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims. The word “comprising” inthe claims may be replaced by “consisting essentially of” or with“consisting of,” according to standard practice in patent law.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic of an exemplary biochemical pathway leading tobiosynthesis of (S) 2-aminopimelate using meso-2,6-diaminopimelate as acentral metabolite.

FIG. 2 is a schematic of an exemplary biochemical pathway leading to thebiosynthesis of (R) 2-aminopimelate using meso-2,6-diaminopimelate as acentral metabolite.

FIG. 3 is a schematic of exemplary biochemical pathways leading toadipic acid using either (S) 2-aminopimelate or (R) 2-aminopimelate as acentral precursor.

FIG. 4 is a schematic of exemplary biochemical pathways leading to6-aminohexanoic acid using either (S) 2-aminopimelate, (R)2-aminopimelate or adipate semialdehyde as a central precursor. FIG. 4also contains a schematic of an exemplary biochemical pathway tocaprolactam from 6-aminohexanoic acid.

FIG. 5 is a schematic of exemplary biochemical pathways leading tohexamethylenediamine using 6-aminohexanoic acid or adipate semialdehydeas a central precursor.

FIG. 6 is a schematic of an exemplary biochemical pathway leading tohexamethylenediamine using 6-aminohexanoic acid as a central precursor.

FIG. 7 is a schematic of an exemplary biochemical pathway leading tohexamethylenediamine using 6-hydroxyhexanoic acid as a centralprecursor.

FIG. 8 is a schematic of (i) exemplary biochemical pathways leading to6-hydroxyhexanoic acid using either (S) 2-aminopimelate or (R)2-aminopimelate as a central precursor and (ii) exemplary biochemicalpathways leading to 1,6-hexanediol using 6-hydroxyhexanoic acid as acentral precursor.

FIG. 9 is a bar graph summarizing the change in absorbance at 340 nmafter 20 minutes, which is a measure of the consumption of NADPH andactivity of carboxylate reductases relative to the enzyme only controls(no substrate).

FIG. 10 is a bar graph of the change in absorbance at 340 nm after 20minutes, which is a measure of the consumption of NADPH and the activityof carboxylate reductases for converting adipate to adipate semialdehyderelative to the empty vector control.

FIG. 11 is a bar graph of the change in absorbance at 340 nm after 20minutes, which is a measure of the consumption of NADPH and the activityof carboxylate reductases for converting 6-hydroxhexanoate to6-hydroxhexanal relative to the empty vector control.

FIG. 12 is a bar graph of the change in absorbance at 340 nm after 20minutes, which is a measure of the consumption of NADPH and the activityof carboxylate reductases for converting N6-acetyl-6-aminohexanoate toN6-acetyl-6-aminohexanal relative to the empty vector control.

FIG. 13 is a bar graph of the change in absorbance at 340 nm after 20minutes, which is a measure of the consumption of NADPH and activity ofcarboxylate reductases for converting adipate semialdehyde to hexanedialrelative to the empty vector control.

FIG. 14 is a bar graph summarizing the percent conversion after 4 hoursof pyruvate to L-alanine (mol/mol) as a measure of the ω-transaminaseactivity of the enzyme only controls (no substrate).

FIG. 15 is a bar graph of the percent conversion after 24 hours ofpyruvate to L-alanine (mol/mol) as a measure of the ω-transaminaseactivity for converting 6-aminohexanoate to adipate semialdehyderelative to the empty vector control.

FIG. 16 is a bar graph of the percent conversion after 4 hours ofL-alanine to pyruvate (mol/mol) as a measure of the ω-transaminaseactivity for converting adipate semialdehyde to 6-aminohexanoaterelative to the empty vector control.

FIG. 17 is a bar graph of the percent conversion after 4 hours ofpyruvate to L-alanine (mol/mol) as a measure of the ω-transaminaseactivity for converting hexamethylenediamine to 6-aminohexanal relativeto the empty vector control.

FIG. 18 is a bar graph of the percent conversion after 4 hours ofpyruvate to L-alanine (mol/mol) as a measure of the ω-transaminaseactivity for converting N6-acetyl-1,6-diaminohexane toN6-acetyl-6-aminohexanal relative to the empty vector control.

FIG. 19 is a bar graph of the percent conversion after 4 hours ofpyruvate to L-alanine (mol/mol) as a measure of the o-transaminaseactivity for converting 6-aminohexanol to 6-oxohexanol relative to theempty vector control.

FIGS. 20A-20K contains the amino acid sequences of a Lactobacillusbrevis thioesterase (see GenBank Accession No. ABJ63754.1, SEQ ID NO:1), an Lactobacillus plantarum thioesterase (see GenBank Accession No.CCC78182.1, SEQ ID NO: 2), Mycobacterium marimum carboxylate reductase(see Genbank Accession No. ACC40567.1, SEQ ID NO: 3), a Mycobacteriumsmegmatis carboxylate reductase (see Genbank Accession No. ABK71854.1,SEQ ID NO: 4), a Segniliparus rugosus carboxylate reductase (see GenbankAccession No. EFV11917.1, SEQ ID NO: 5), a Mycobacterium massiliensecarboxylate reductase (see Genbank Accession No. EIV11143.1, SEQ ID NO:6), a Segniliparus rotundus carboxylate reductase (see Genbank AccessionNo. ADG98140.1, SEQ ID NO: 7), a Chromobacterium violaceumω-transaminase (see Genbank Accession No. AAQ59697.1, SEQ ID NO: 8), aPseudomonas aeruginosa ω-transaminase (see Genbank Accession No.AAG08191.1, SEQ ID NO: 9), a Pseudomonas syringae ω-transaminase (seeGenbank Accession No. AAY39893.1, SEQ ID NO: 10), a Rhodobactersphaeroides ω-transaminase (see Genbank Accession No. ARA81135.1, SEQ IDNO: 11), an Escherichia coli ω-transaminase (see Genbank Accession No.AAA57874.1, SEQ ID NO: 12), a Vibrio fluvialis ω-transaminase (seeGenbank Accession No. AEA39183.1, SEQ ID NO: 13), a Bacillus subtilisphosphopantetheinyl transferase (see Genbank Accession No. CAA44858.1,SEQ ID NO:14), a Nocardia sp. NRRL 5646 phosphopantetheinyl transferase(see Genbank Accession No. AB183656.1, SEQ ID NO:15), a Bacillussubtilis enoate reductase (see Genbank Accession No. BAA12619.1, SEQ IDNO: 16), a Pseudomonas putida

enoate reductase (see Genbank Accession No. AAN66878.1., SEQ ID NO: 17),a Kluyveromyces lactis enoate reductase (see Genbank Accession No.AAA9881.5A, SEQ ID NO: 18), a Lactobacillus casei enoate reductase (seeGenbank Accession No. AGP69310,1, SEQ ID NO: 19), a Saccharomycespastorianus enoate reductase (see Genbank Accession No. CAA37666.1, SEQID NO: 20), a Thermaanaerobacter pseudethanolicus enoate reductase (seeGenbank Accession No. ABY93685.1, SEQ ID NO: 21), an Enterobactercloacae enoate reductase (see Genbank Accession No. AAB38683.1, SEQ IDNO: 22), a Fusobacterium nucleatum ammonia lyase (see Genbank AccessionNo. AAL93968.1, SEQ ID NO: 23), an Acidaminococcus fermentans2-hydroxyglutaryl-CoA dehydratase activator (see Genbank Accession No.CAA42196A, SEQ ID NO: 24), a Clostridium symbiosum 2-hydroxyglutaryl-CoAdehydratase (see Genbank Accession No. AAD31677.1 & AAD31675.1, SEQ IDNO: 25), a Bacillus subtilis aminomutase (see Genbank Accession No.AAB72069.1, SEQ ID NO: 26), a Peptoclostridium difficile2-Hydroxyisocaproyl-CoA dehydratase activator (see Genbank Accession No.AAV40818.1, SEQ ID NO: 27), a Peptodostridium difficile2-Hydroxyisocaproyl-CoA dehydratase (see Genbank Accession No.AAV40819.1 & AAV40820.1, SEQ ID NO: 28), an Escherichia coli glutamatedecarboxylase (see Genbank Accession No. AAA23833.1, SEQ ID NO: 29), anEscherichia coli lysine decarboxylase (see Genbank Accession No.AAA23536.1, SEQ ID NO: 30), an Escherichia coli ornithine decarboxylase(see Genbank Accession No. AAA62785.1, SEQ ID NO: 31), an Escherichiacoli lysine decarboxylase (see Genbank Accession No. BAA21656.1, SEQ IDNO: 32), an Escherichia coli diaminopimelate decarboxylase (see GenbankAccession No. AAA83861.1., SEQ ID NO: 33), and a Salmonella typhimuriumindole-3-pyruvate decarboxylase (see Genbank Accession NO. CAC48239.1,SEQ ID: 34).

DETAILED DESCRIPTION

Described herein are enzymes, non-natural pathways, cultivationstrategies, feedstocks, host microorganisms and attenuations to thehost's biochemical network, which generates a seven carbon chainaliphatic backbone from central metabolites which can be decarboxylatedto a six carbon aliphatic backbone into which one or two terminalfunctional groups may be formed leading to the synthesis of adipic acid,adipate semialdehyde, caprolactam, 6-aminohexanoic acid,6-hydroxyhexanoic acid, hexamethylenediamine or 1,6-hexanediol (referredto as “C6 building blocks” herein). As used herein, the term “centralprecursor” is used to denote any metabolite in any metabolic pathwayshown herein leading to the synthesis of one or more C6 building blocks.The term “central metabolite” is used herein to denote a metabolite thatis produced in all microorganisms to support growth.

Host microorganisms described herein can include endogenous pathwaysthat can be manipulated such that one or more C6 building blocks can beproduced. In an endogenous pathway, the host microorganism naturallyexpresses all of the enzymes catalyzing the reactions within thepathway. A host microorganism containing an engineered pathway does notnaturally express all of the enzymes catalyzing the reactions within thepathway but has been engineered such that all of the enzymes within thepathway are expressed in the host. Within an engineered pathway, theenzymes can be from a single source, i.e., from one species, or can befrom multiple sources, i.e., different species or genera. Nucleic acidsencoding the enzymes described herein have been identified from variousorganisms and are readily available in publicly available databases suchas GenBank or EMBL. Engineered hosts can naturally express none or some(e.g., one or more, two or more, three or more, four or more, five ormore, or six or more) of the enzymes of the pathways described herein.Thus, a pathway within an o engineered host can include all exogenousenzymes, or can include both endogenous and exogenous enzymes.Endogenous genes of the engineered hosts also can be disrupted toprevent the formation of undesirable metabolites or prevent the loss ofintermediates in the pathway through other enzymes acting on suchintermediates. Engineered hosts can be referred to as recombinant hostsor recombinant host cells. Thus, as described herein recombinant hostscan include nucleic acids encoding one or more of a dehydrogenase,decarboxylase, reductase, dehydratase, CoA-transferase, thioesterase,hydrolase, ammonia lyase, mutase, synthase, aminotransferase, ortransaminase as described in more detail below.

The term “exogenous” as used herein with reference to a nucleic acid (ora protein) and a host refers to a nucleic acid that does not occur in(and cannot be obtained from) a cell of that particular type as it isfound in nature or a protein encoded by such a nucleic acid. Thus, anon-naturally-occurring nucleic acid is considered to be exogenous to ahost once in the host. It is important to note thatnon-naturally-occurring nucleic acids can contain nucleic acidsubsequences or fragments of nucleic acid sequences that are found innature provided the nucleic acid as a whole does not exist in nature.For example, a nucleic acid molecule containing a genomic DNA sequencewithin an expression vector is non-naturally-occurring nucleic acid, andthus is exogenous to a host cell once introduced into the host, sincethat nucleic acid molecule as a whole (genomic DNA plus vector DNA) doesnot exist in nature. Thus, any vector, autonomously replicating plasmid,or virus (e.g., retrovirus, adenovirus, or herpes virus) that as a wholedoes not exist in nature is considered to be non-naturally-occurringnucleic acid. It follows that genomic DNA fragments produced by PCR orrestriction endonuclease treatment as well as cDNAs are considered to henon-naturally-occurring nucleic acid since they exist as separatemolecules not found in nature. It also follows that any nucleic acidcontaining a promoter sequence and polypeptide-encoding sequence (e.g.,cDNA or genomic DNA) in an arrangement not found in nature isnon-naturally-occurring nucleic acid. A nucleic acid that isnaturally-occurring can be exogenous to a particular host microorganism.For example, an entire chromosome isolated from a cell of yeast x is anexogenous nucleic acid with respect to a cell of yeast y once that ochromosome is introduced into a cell of yeast y.

In contrast, the term “endogenous” as used herein with reference to anucleic acid (e.g., a gene) (or a protein) and a host refers to anucleic acid (or protein) that does occur in (and can be obtained from)that particular host as it is found in nature. Moreover, a cell“endogenously expressing” a nucleic acid (or protein) expresses thatnucleic acid (or protein) as does a host of the same particular type asit is found in nature. Moreover, a host “endogenously producing” or that“endogenously produces” a nucleic acid, protein, or other compoundproduces that nucleic acid, protein, or compound as does a host of thesame particular type as it is found in nature.

In some embodiments, depending on the host and the compounds produced bythe host, one or more of the following polypeptides having2-hydroxyacyl-CoA dehydratase activity, mutase activity, ammonia lyaseactivity, and enoate reductase activity may be expressed in the host inaddition to one or more of: a polypeptide having α-oxoacid decarboxylaseactivity, a polypeptide having α-aminoacid decarboxylase activity, apolypeptide having synthase activity, a polypeptide having the activityof a dehydrogenase complex, a polypeptide having diaminopimelatedehydrogenase activity, a polypeptide having (R)-2-hydroxyisocaproatedehydrogenase activity, a polypeptide having (R)-2-hydroxyglutaratedehydrogenase activity, a polypeptide having glutaconate CoA-transferaseactivity, a polypeptide having 2-hydroxyisocaproyl-CoA dehydrataseactivity, a polypeptide having (R)-2-hydroxyglunyl-CoA dehydrataseactivity, a polypeptide having carboxylate reductase activity, apolypeptide having aldehyde dehydrogenase activity, a polypeptide havinglysine 2,3-aminomutase activity, a polypeptide having succinate-CoAligase activity, a polypeptide having 3-aminobutyryl-CoA ammonia lyaseactivity, a polypeptide having thioesterase activity, a polypeptidehaving CoA-transferase activity, a polypeptide havingalpha-aminotransferase activity, a polypeptide havingbranch-chain-2-oxoacid decarboxylase activity, a polypeptide havingacetolactate synthase activity, a polypeptide having aldehydedehydrogenase activity, a polypeptide having hydrolase activity, apolypeptide having ω-transaminase activity, a polypeptide havingN-acetyltransferase activity, a polypeptide having lysineN-acetyltransferase activity, or a polypeptide having alcoholdehydrogenase activity. In recombinant hosts expressing a carboxylatereductase, a phosphopantetheinyl transferase also can be expressed as itenhances activity of the carboxylase reductase.

For example, a recombinant host can include at least one exogenouspolypeptide having an activity selected from the group consisting of2-hydroxyacyl-CoA dehydratase activity, mutase activity, ammonia lyaseactivity, and enoate reductase activity and produce 2-aminopimelate from2,6-diaminopimelate.

For example, a host can include an exogenous polypeptide having2-hydroxyacyl-CoA dehydratase activity and an exogenous polypeptidehaving enoate reductase activity and produce 2-aminopimelate (e.g.,(S)-aminopimelate). Such a host further can include at least onepolypeptide having an activity selected from the group consisting ofdiaminopimelate dehydrogenase activity, 2-hydroxycarboxylatedehydrogenase activity, CoA-transferase activity, 2-hydroxyaciddehydratase activity, and carboxylate reductase activity. See, e.g.,FIG. 1.

For example, a recombinant host can include (i) an exogenous polypeptidehaving diaminopimelate dehydrogenase activity classified, for example,under EC 1.4.1.16, (ii) an exogenous polypeptide having2-hydroxyisocaproate dehydrogenase activity or an exogenous polypeptidehaving (R)-2-hydroxyglutarate dehydrogenase activity classified, forexample, under EC 1.1.1.- such as EC 1.1.1.337, (iii) an exogenouspolypeptide having glutaconate CoA-transferase activity classified, forexample, under EC 2.8.3.12, (iv) an exogenous polypeptide having2-hydroxyisocaproyl-CoA dehydratase activity or a polypeptide having2-hydroxyglutryl-CoA dehydratase activity classified, for example, underEC 4.2.1.-, (v) an exogenous polypeptide having carboxylase reductaseactivity classified, for example, under EC 1.2.99.6, (vi) an exogenouspolypeptide having enoate reductase activity classified, for example,under EC 1.3.1.31 or EC 1.3.99.1, (vii) or an exogenous polypeptidehaving aldehyde dehydrogenase activity classified, for example, under EC1.2.1.—such as EC 1.2.1.3 and produce (S) 2-aminopimelate. See, FIG. 1.

For example, a recombinant host can include an exogenous polypeptidehaving mutase activity, an exogenous polypeptide having ammonia lyaseactivity, and an exogenous polypeptide having enoate reductase activityand produce 2-aminopimelate (e.g., (R)-aminopimelate). Such a hostfurther can include at least one polypeptide o having an activityselected from the group consisting of CoA ligase activity,CoA-transferase activity, carboxylate reductase activity, and aldehydedehydrogenase activity. See, FIG. 2.

For example, a recombinant host can include (i) an exogenous polypeptidehaving lysine 2,3-aminomutase activity classified, for example, under EC5.4.3.2, (ii) an exogenous polypeptide having succinate-CoA ligaseactivity classified, for example, under EC 6.2.1.5 or a polypeptidehaving CoA-transferase activity classified, for example, under EC2.8.3.-, (iii) an exogenous polypeptide having 3-aminobutyryl-CoAammonia lyase activity classified, for example, under EC 4.3.1.14, (iv)an exogenous polypeptide having thioesterase activity classified, forexample, under EC 3.1.2.- or polypeptide having CoA-transferase activityclassified, for example, under EC 2.8.3.-, (v) an exogenous polypeptidehaving carboxylate reductase activity classified, for example, under EC1.2.99.6, (vi) an exogenous polypeptide having enoate reductase activityclassified, for example, under EC 1.3.1.31 or EC 1.6.99.1 or (vii) apolypeptide having aldehyde dehydrogenase activity classified, forexample, under EC 1.2.1.—such as EC 1.2.1.3 and produce (R)2-aminopimelate.

A recombinant host producing 2-aminopimelate also can include at leastone exogenous polypeptide having an activity selected from the groupconsisting of α-oxoacid decarboxylase activity classified under ECα-aminoacid decarboxylase activity classified under EC 4.1.1.-, synthaseactivity, and activity of a dehydrogenase complex. See, e.g., FIG. 3 andFIG. 4.

In some embodiments, a recombinant host producing 2-aminopimelate caninclude an exogenous polypeptide having 2-oxoacid decarboxylase activityclassified, for example, under EC 4.1.1.—such as EC 4.1.1.43, EC4.1.1.71, EC 4.1.1.72 or EC 4.1.1.74 or an exogenous polypeptide havingacetolactate synthase activity classified, for example, under EC 2.2.1.6and produce adipic acid. See, FIG. 3.

For example, a recombinant host producing 2-aminopimelate can include(i) an exogenous polypeptide having 2-oxoacid decarboxylase activityclassified, for example, under EC 4.1.1.—such as EC 4.1.1.43, EC4.1.1.71, EC 4.1.1.72 or EC 4.1174 or an exogenous polypeptide havingacetolactate synthase activity classified, for example, under EC 2.2.1.6and (ii) an exogenous polypeptide having α-aminotransferase activityclassified, for example, under EC 2.6.1.—such as EC 2.6.1.39, EC2.6.1.42 or EC 2.6.1.21 and produce adipate semialdehyde or adipic acid.See, FIG. 3.

For example, a recombinant host producing 2-aminopimelate can include(i) an exogenous polypeptide having 2-oxoacid decarboxylase activityclassified, for example, under EC 4.1.1.—such as EC 4.1.1.43, EC4.1.1.71, EC 4.1.1.72 or EC 4.1.1.74 or an exogenous polypeptide havingacetolactate synthase activity classified, for example, under EC2.2.1.6, (ii) an exogenous polypeptide having α-aminotransferaseactivity classified, for example, under EC 2.6.1.—such as EC 2.6.1.39,EC 2.6.1.42 or EC 2.6.1.21, and (iii) an exogenous polypeptide havingaldehyde dehydrogenase activity classified, for example, under EC1.2.1.—such as EC 1.2.1.3, EC 1.2.1.16, EC 1.2.1.20, EC 1.2.1.63 or EC1.2.1.79 and produce adipic acid. See, FIG. 3.

In some embodiments, a recombinant host producing 2-aminopimelate caninclude an exogenous polypeptide having acetolactate synthase activityclassified, for example, under EC 2.2.1.6 and produce adipic acid. See,FIG. 3.

For example, a recombinant host producing 2-aminopimelate can include(i) an exogenous polypeptide having acetolactate synthase activityclassified, for example, under EC 2.2.1.6 and an exogenous polypeptidehaving α-aminotransferase activity classified, for example, under EC2.6.1.—such as EC 2.6.1.39, EC 2.6.1.42 or EC 2.6.1.21, and produceadipic acid. See, FIG. 3.

For example, a recombinant host producing 2-aminopimelate can include anexogenous polypeptide having acetolactate synthase activity classified,for example, under EC 2.2.1.6, an exogenous polypeptide havingalpha-aminotransferase activity classified, for example, under EC2.6.1.—such as EC 2.6.1.39, EC 2.6.1.42 or EC 2.6.1.21, and an exogenouspolypeptide having aldehyde dehydrogenase activity classified, forexample, under EC 1.2.1.—such as EC 1.2.1.3, EC 1.2.1.16, EC 1.2.1.20,EC 1.2.1.63 or EC 1.2.1.79 and produce adipic acid. See, FIG. 3.

In some embodiments, a recombinant host producing 2-aminopimelate caninclude an exogenous dehydrogenase complex comprised of enzymeactivities classified, for example, EC 1.2.4.2, EC 1.8.1.4 or EC2.3.1.61 and produce adipic acid. See, FIG. 3.

For example, a recombinant host producing 2-aminopimelate can include(i) an exogenous dehydrogenase complex comprised of enzyme activitiesclassified, for example, EC 1.2.4.2, EC 1.8.1.4 or EC 2.3.1.61 and (ii)an exogenous polypeptide having α-aminotransferase activity classified,for example, under EC 2.6.1.—such as EC 2,6.1.39, EC 2,6.1.42 or EC2,6.1.21 and produce adipic acid. See, FIG. 3.

For example, a recombinant host producing 2-aminopimelate can include anexogenous dehydrogenase complex comprised of enzyme activitiesclassified, for example, EC 1.2.4.2, EC 1.8.1.4 or EC 2.3.1.61 and anexogenous polypeptide having thioesterase activity classified, forexample, under EC 3.1.2.—and produce adipic acid. See, FIG. 3.

For example, a recombinant host producing 2-aminopimelate can include anexogenous dehydrogenase complex and an exogenous polypeptide havingglutaconate CoA-transferase activity classified, for example, under EC2.8.3.12 or an exogenous polypeptide having succinate CoA-ligaseactivity classified, for example, under EC 6.2.1.5 and produce adipicacid. See, FIG. 3.

For example, a recombinant host producing 2-aminopimelate can include(i) an exogenous dehydrogenase complex comprised of enzyme activitiesclassified, for example, EC 1.2.4.2, EC 1.8.1.4 or EC 2.3.1,61, (ii) anexogenous polypeptide having alpha-aminotransferase activity classified,for example, under EC 2.6.1.—such as EC 2.6.1.39, EC 2.6.1.42 or EC2.6.1.21, and (iii) an exogenous polypeptide having thioesteraseactivity classified, for example, under EC 3.1.2.-, a polypeptide havingCoA-ligase activity classified, for example, under EC 6.2.1.5 or apolypeptide having CoA-transferase activity classified, for example,under EC 2.8.3.12 and produce adipic acid. See, FIG. 3.

For example, a recombinant host producing 2-aminopimelate can include anexogenous dehydrogenase complex, an exogenous polypeptide havingalpha-aminotransferase activity, and an exogenous polypeptide havinggintaconate CoA-transferase activity or an exogenous polypeptide havingsuccinelle CoA-ligase activity and produce adipic acid. See, FIG. 3.

In some embodiments, a recombinant host producing (S)-2-aminopimelatecan include a polypeptide having decarboxylase activity classified, forexample, under EC 4.1.1.—such as EC 4.1.1.15, EC 4.1.1.17, EC 4.1.1.18,EC 4.1.1.19 and produce 6-aminohexanoic acid, which can be converted tocaprolactam using an exogenous polypeptide having amidohydrolaseactivity (classified, for example, under EC 3.5.2,-). See, FIG. 4.

In some embodiments, a recombinant host producing (R)-2-aminopimelatecan include a polypeptide having decarboxylase activity classified, forexample, under EC 4.1.1.- such as EC 4.1.1.20 and produce6-aminohexanoic acid from (R)-2-aminopimelate, which can be converted tocaprolactam using an exogenous polypeptide having hydrolase activity(classified, for example, under EC 3.5.2.4 See, FIG. 4.

A recombinant host producing 2-aminopimelate can include (i) anexogenous polypeptide having α-aminotransferase activity classified, forexample, under EC 2.6.1—such as EC 2.6.1.21, EC 2.6.1.39 or EC 2.6.1.42(ii) an exogenous polypeptide having decarboxylase activity classified,for example, under EC 4.1.1.—such as EC 4.1,1.43, EC 4.1.1.71, EC4.1.1.71 or EC 4.1.1.74 or a polypeptide having acetolactate synthaseactivity classified, for example, under EC 2.2.1.6 and (iii) anexogenous polypeptide having ω-transaminase activity classified, forexample, under EC 2.6.1.—such EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.48, EC2.6.1.29, or EC 2.6.1.82 and produce 6-aminohexanoic acid. See, FIG. 4.

A recombinant host producing 6-aminohexanoic acid can further include(i) an exogenous polypeptide having carboxylate reductase activityclassified, for example, under EC 1.2.99.6 (ii) an exogenous polypeptidehaving ω-transaminase activity classified, for example, under EC2.6.1.—such as EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.48, EC 2.6.1.29, or EC2.6.1.82 and produce hexamethylenediamine. See, FIG. 5.

A recombinant host producing 2-aminopimelate can include (i) anexogenous polypeptide having α-aminotransferase activity classified, forexample, under EC 2.6.1.39 or EC 2.6.1.42, (ii) classified, for example,under EC 4.1.1.—such as EC 4.1.1.43, EC 4.1.1.71, EC 4.1.1.71 or EC4.1.1.74 or a polypeptide having acetolactate synthase activityclassified, for example, under EC 2.2.1.6, (iii) a polypeptide havingcarboxylate reductase activity classified, for example, under EC1.2.99.6 and (iv) exogenous polypeptide having ω-transaminase activityclassified, for example, under EC 2.6.1.—such EC 2.6.1.18, EC 2.6.1.19,EC 2.6.1.48, EC 2.6.1.29, or EC 2.6.1.82 and producehexamethylenediamine. See, FIG. 5.

A recombinant host producing 6-aminohexanoic acid can further include(i) an exogenous polypeptide having N-acetyltransferase activityclassified, for example, under EC 2.3.1.32 (ii) a polypeptide havingcarboxylate reductase activity classified, for example, under EC1.2.99.6, (iii) a polypeptide having ω-transaminase activity classified,for example, under EC 2.6.1.—such as EC 2.6.1.18, EC 2.6.1.19, or EC2.6.1.48, EC 2.6.1.29, or EC 2.6.1.82 and (iv) and a polypeptide havingdeacylase activity classified, for example, under EC 3.5.1.17 andproduce hexamethylenediamine. See, FIG. 6.

In some embodiments, a recombinant host can include a polypeptide havingα-aminotransferase activity classified, for example, under EC2.6.1.—such as EC 2.6.1.39, EC 2.6.1.42 or EC 2.6.1.21 and produce6-hydroxyhexanoic acid. See, FIG. 7.

For example, a recombinant host can include (i) a polypeptide havingα-aminotransferase activity classified, for example, under EC2.6.1.—such as EC 2.6.1.39, EC 2.6.1.42 or EC 2.6.1.21 and (ii) anexogenous polypeptide having 2-oxoacid decarboxylase activityclassified, for example, under EC 4.1.1.—such as EC 4.1.1.43, EC4.1.1.71, EC 4.1.1.72 or EC 4.1.1.74 or an exogenous polypeptide havingacetolactate synthase activity classified, for example, under EC 2.2.1.6and produce 6-hydroxyhexanoic acid, See, FIG. 8.

For example, a recombinant host can include (i) a polypeptide havingα-aminotransferase activity classified, for example, under EC2.6.1.—such as EC 2.6.1.39, EC 2.6.1.42 or EC 2.6.1.21 and (ii) anexogenous polypeptide having 2-oxoacid decarboxylase activityclassified, for example, under EC 4.1.1.—such as EC 4.1.1.43, EC4.1.1.71, EC 4.1.1.72 or EC 4.1.1.74 or an exogenous polypeptide havingacetolactate synthase activity classified, for example, under EC2.2.1.6, (iii) and a polypeptide having alcohol dehydrogenase activityclassified, for example, under EC 1.1.1.—such as EC 1.1.1.2 or EC1.1.1.258 and produce 6-hydroxyhexanoic acid. See, FIG. 8.

A recombinant host producing 6-hydroxyhexanoic acid can further include(i) a polypeptide having carboxylate reductase activity classified, forexample, under EC 1.2.99.6, (ii) a polypeptide having ortransaminaseactivity classified, for example, under EC 2.6.1.—such as EC 2.6.1.18,EC 2.6.1.19, or EC 2.6.1.48, EC 2.6.1.29, or EC 2.6.1.82, and (iii) apolypeptide having alcohol dehydrogenase activity classified, forexample, under EC 1.1.1.—such as EC 1.1.1.1 and producehexamethylenediamine. See, FIG. 7.

A recombinant host producing 6-hydroxyhexanoic acid can further include(i) an exogenous polypeptide having carboxylate reductase activityclassified, for example, under EC 1.2.99.6 and (ii) an exogenouspolypeptide having alcohol dehydrogenase activity classified, forexample, under EC 1.1.1.—such as EC 1.1.1.1, EC 1.1.1.2, EC 1.1.1.21 orEC 1.1.1.184 and produce 1,6 hexanediol. See, FIG. 8.

Any of the enzymes described herein that can be used for production ofone or more C6 building blocks can have at least 70% sequence identity(homology) (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of thecorresponding wild-type enzyme. It will be appreciated that the sequenceidentity can be determined on the basis of the mature enzyme (e.g., withany signal sequence removed) or on the basis of the immature enzyme(e.g., with any signal sequence included). It also will be appreciatedthat the initial methionine residue may or may not be present on any ofthe enzyme sequences described herein.

Any of the enzymes described herein that can be used for production ofone or more C6 building blocks can have at least 70% sequence identity(homology) (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or100%) to the amino acid sequence of the corresponding wild-type enzyme.For example, a thioesterase described herein can have at least 70%sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 95%,97%, 98%, 99%, or 100%) to the amino acid sequence of a Lactobacillusbrevis thioesterase (see GenBank Accession No. ABJ63754.1, SEQ ID NO: 1)or to the amino acid sequence of a Lactobacillus plantarum thioesterase(see GenBank Accession No. CCC78182.1, SEQ ID NO: 2). See FIG. 20A.

For example, a carboxylate reductase described herein can have at least70% sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%,95%, 97%, 98%, 99%, or 100%) to the amino acid sequence of aMycobacterium marimum (see Genbank Accession No. ACC40567.1, SEQ ID NO:3), a Mycobacterium smegmatis (see Genbank Accession No. ABK71854.1, SEQID NO: 4), a Segniliparus rugosus (see Genbank Accession No. EFV11917.1,SEQ ID NO: 5), a Mycobacterium massiliense (see Genbank Accession No.EIV11143.1, SEQ ID NO: 6), or a Segniliparus rotundus (see GenbankAccession No. ADG98140.1, SEQ ID NO: 7) carboxylate reductase. See,FIGS. 20A-20E.

For example, a ω-transaminase described herein can have at least 70%sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 95%,97%, 98%, 99%, or 100%) to the amino acid sequence of a Chromobacteriumviolaceum (see Genbank Accession No. AAQ59697.1, SEQ ID NO: 8), aPseudomonas aeruginosa (see Genbank Accession No. AAG08191.1, SEQ ID NO:9), a Pseudomonas syringae (see Genbank Accession No. AAY39893.1, SEQ IDNO: 10), a Rhodobacter sphaeroides (see Genbank Accession No.ABA81135.1, SEQ ID NO: 11), an Escherichia coli (see Genbank AccessionNo. AAA57874.1, SEQ ID NO: 12), or a Vibrio fluvialis (see GenbankAccession No. AEA39183.1, SEQ ID NO: 13) ω-transaminase. Some of theseω-transaminases are diamine ω-transaminases. See, FIG. 20E and FIG. 20F.

For example, a phosphopantetheinyl transferase described herein can haveat least 70% sequence identity (homology) (e.g., at least 75%, 80%, 85%,90%, 95%, 97%, 98%, 99%, or 100%) to the amino acid sequence of aBacillus subtilis phosphopantetheinyl transferase (see Genbank AccessionNo. CAA44858,1, SEQ ID NO: 14) or a Nocardia sp. NRRL 5646phosphopantetheinyl transferase (see Genbank Accession No. ABI83656.1,SEQ ID NO:15), See, FIG. 20F and FIG. 20G.

For example, an enoate reductase described herein can have at least 70%sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 95%,9.7%, 98%, 99%, or 100%) to the amino acid sequence of a Bacillussubtilis enoate reductase (see Genbank Accession No. BAA12619.1, SEQ IDNO: 16), a Pseudomonas putida enoate reductase (see Genbank AccessionNo. AAN66878,1, SEQ ID NO: 17), a Kluyveromyces lactis enoate reductase(see Genbank Accession No. AAA98815.1, SEQ ID NO: 18), a Lactobacilluscasei enoate reductase (see Genbank Accession No. AGP69310.1, SEQ ID NO:19), a Saccharomyces pastoriamus enoate reductase (see Genbank AccessionNo. CAA37666.1, SEQ ID NO: 20), a Thermoanaerobacter pseudethanolicusenoate reductase (see Genbank Accession No. ABY93685.1, SEQ ID NO: 21),a Enterobacter cloacae enoate reductase (see Genbank Accession No.AAB38683.1, SEQ ID NO: 22).

For example, an ammonia lyase described herein can have at least 70%sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 95%,97%, 98%, 99%, or 100%) to the amino acid sequence of a Fusobacterhunnucleatum ammonia lyase (see Genbank Accession No. AAL93968.1, SEQ IDNO: 23).

For example, a dehydratase activator described herein can have at least70% sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%,95%, 97%, 98%, 99%, or 100%) to the amino acid sequence of anAcidaminococcus fermentans 2-hydroxyglutaryl-CoA dehydratase activator(see Genbank Accession No. CAA.42196.1, SEQ ID NO: 24) or aPeptoclostridium difficile 2-Hydroxyisocaproyl-CoA dehydratase activator(see Genbank Accession No. AAV40818.1, SEQ ID NO: 27).

For example, a 2-hydraryacyl-CoA dehydratase described herein can haveat least 70% sequence identity (homology) (e.g., at least 75%, 80%, 85%,90%, 95%, 97%, 98%, 99%, or 100%) to the amino acid sequence of aClostridium symbiosum 2-hydroxyglutaryl-CoA dehydratase (see GenbankAccession No. AAD31677.1 & AAD31675.1, SEQ ID NO: 25), or aPeptoclostrillum difficile 2-Hydroxyisocaproyl-CoA dehydratase (seeGenbank Accession No. AAV40819.1 & AAV40820.1, SEQ ID NO: 28).

For example, an aminomutase described herein can have at least 70%sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 95%,97%, 98%, 99%, or 100%) to the amino acid sequence of a Bacillussubtilis aminomutase (see Genbank Accession No. AAB72069.1, SEQ ID NO:26).

For example, a decarboxylase described herein can have at least 70%sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 95%,97%, 98%, 99%, or 100%) to the amino acid sequence of an Escherichiacoli glutamate decarboxylase (see Genbank Accession No. AAA23833.1, SEQID NO: 29), an Escherichia coli lysine decarboxylase (see GenbankAccession No. AAA23536.1, SEQ ID NO: 30), an Escherichia coli ornithinedecarboxylase (see Genbank Accession No. AAA62785.1, SEQ ID NO: 31), anEscherichia coli lysine decarboxylase (see Genbank Accession No.BAA21656,1, SEQ ID NO: 32), an Escherichia coli diaminopimelatedecarboxylase (see Genbank Accession No. AAA83861.1, SEQ ID NO: 33), aSalmonella typhimurium indole-3-pyruvate decarboxylase (see GenbankAccession No. CAC48239,1, SEQ ID NO: 34).

The percent identity (homology) between two amino acid sequences can bedetermined as follows. First, the amino acid sequences are aligned usingthe BLAST 2 Sequences (Bl2seq) program from the stand-alone version ofBLASTZ containing BLASTP version 2.0.14. This stand-alone version ofBLASTZ can be obtained from Fish & Richardson's web site (e.g.,www.fr.com/blast/) or the U.S. government's National Center forBiotechnology Information web site (www.ncbi.nlm.nih.gov). Instructionsexplaining how to use the Bl2seq program can be found in the readme fileaccompanying BLASTZ. Bl2seq performs a comparison between two amino acidsequences using the BLASTP algorithm. To compare two amino acidsequences, the options of Bl2seq are set as follows: -i is set to a filecontaining the first amino acid sequence to be compared (e.g.,C:\seq1.txt); -j is set to a file containing the second amino acidsequence to be compared (e.g., C:\seq2.txt); -p is set to blastp; -o isset to any desired file name (e.g., C:\output.txt); and all otheroptions are left at their default setting. For example, the followingcommand can be used to generate an output file containing a comparisonbetween two amino acid sequences: C:\Bl2seq -i c:\seq1.txt -jc:\seq2.txt -p blastp -o c:\output.txt. if the two compared sequencesshare homology (identity), then the designated output file will presentthose regions of homology as aligned sequences. If the two comparedsequences do not share homology (identity), then the designated outputfile will not present aligned sequences. Similar procedures can befollowing for nucleic acid sequences except that blastn is used.

Once aligned, the number of matches is determined by counting the numberof positions where an identical amino acid residue is presented in bothsequences. The o percent identity (homology) is determined by dividingthe number of matches by the length of the full-length polypeptide aminoacid sequence followed by multiplying the resulting value by 100. It isnoted that the percent identity (homology) value is rounded to thenearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 is roundeddown to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 is rounded upto 78.2. It also is noted that the length value will always be aninteger.

It will be appreciated that a number of nucleic acids can encode apolypeptide having a particular amino acid sequence. The degeneracy ofthe genetic code is well known to the art; i.e., for many amino acids,there is more than one nucleotide triplet that serves as the codon forthe amino acid. For example, codons in the coding sequence for a givenenzyme can be modified such that optimal expression in a particularspecies (e.g., bacteria or fungus) is obtained, using appropriate codonbias tables for that species.

Functional fragments of any of the enzymes described herein can also beused in the methods of the document. The term “functional fragment” asused herein refers to a peptide fragment of a protein that has at least25% (e.g., at least: 30%; 40%; 50%; 60%; 70%; 75%; 80%; 85%; 90% 95%;98%; 99%; 100%; or even greater than 100%) of the activity of thecorresponding mature, full-length, wild-type protein. The functionalfragment can generally, but not always, be comprised of a continuousregion of the protein, wherein the region has functional activity.

This document also provides (i) functional variants of the enzymes usedin the methods of the document and (ii) functional variants of thefunctional fragments described above. Functional variants of the enzymesand functional fragments can contain additions, deletions, orsubstitutions relative to the corresponding wild-type sequences. Enzymeswith substitutions will generally have not more than 50 (e.g., not morethan one, two, three, four, five, six, seven, eight, nine, ten, 12, 15,20, 25, 30, 35, 40, or 50) amino acid substitutions (e.g., conservativesubstitutions). This applies to any of the enzymes described herein andfunctional fragments. A conservative substitution is a substitution ofone amino acid for another with similar characteristics. Conservativesubstitutions include substitutions within the following groups: valine,alanine and glycine; leucine, valine, and isoleucine; aspartic acid andglutamic acid; asparagine and o glutamine; serine, cysteine, andthreonine; lysine and arginine; and phenylalanine and tyrosine. Thenonpolar hydrophobic amino acids include alanine, leucine, isoleucine,valine, proline, phenylalanine, tryptophan and methionine. The polarneutral amino acids include glycine, serine, threonine, cysteine,tyrosine, asparagine and glutamine. The positively charged (basic) aminoacids include arginine, lysine and histidine. The negatively charged(acidic) amino acids include aspartic acid and glutamic acid. Anysubstitution of one member of the above-mentioned polar, basic or acidicgroups by another member of the same group can be deemed a conservativesubstitution. By contrast, a nonconservative substitution is asubstitution of one amino acid for another with dissimilarcharacteristics.

Deletion variants can lack one, two, three, four, five, six, seven,eight, nine, ten, 11, 12, 13, 14, 15. 16, 17, 18, 19, or 20 amino acidsegments (of two or more amino acids) or non-contiguous single aminoacids. Additions (addition variants) include fusion proteins containing:(a) any of the enzymes described herein or a fragment thereof; and (b)internal or terminal (C or N) irrelevant or heterologous amino acidsequences. In the context of such fusion proteins, the term“heterologous amino acid sequences” refers to an amino acid sequenceother than (a). A heterologous sequence can be, for example a sequenceused for purification of the recombinant protein (e.g., FLAG,polyhistidine (e.g., hexahistidine), hemagglutinin (HA),glutathione-S-transferase (GST), or maltosebinding protein (MBP)).Heterologous sequences also can be proteins useful as detectablemarkers, for example, luciferase, green fluorescent protein (GFP), orchloramphenicol acetyl transferase (CAT). In some embodiments, thefusion protein contains a signal sequence from another protein. Incertain host cells (e.g., yeast host cells), expression and/or secretionof the target protein can be increased through use of a heterologoussignal sequence. In some embodiments, the fusion protein can contain acarrier (e.g., KLH) useful, e.g., in eliciting an immune response forantibody generation) or ER or Golgi apparatus retention signals.Heterologous sequences can be of varying length and in some cases can bea longer sequences than the full-length target proteins to which theheterologous sequences are attached.

In addition, the production of one or more C6 building blocks can beperformed in vitro using the isolated enzymes described herein, using alysate (e.g., a cell lysate) from a host microorganism as a source ofthe enzymes, or using a plurality of lysates from different hostmicroorganisms as the source of the enzymes.

The reactions of the pathways described herein can be performed in oneor more host strains (a) naturally expressing one or more relevantenzymes, (b) genetically engineered to express one or more relevantenzymes, or (c) naturally expressing one or more relevant enzymes andgenetically engineered to express one or more relevant enzymes.Alternatively, relevant enzymes can be extracted from of the above typesof host cells and used in a purified or semi-purified form. Moreover,such extracts include lysates (e.g. cell lysates) that can be used assources of relevant enzymes. In the methods provided by the document,all the steps can be performed in host cells, all the steps can beperformed using extracted enzymes, or some of the steps can be performedin cells and others can be performed using extracted enzymes.

In addition, the production of one or more C6 building blocks can beperformed in vitro using the isolated enzymes described herein, using alysate (e.g., a cell lysate) from a host microorganism as a source ofthe enzymes, or using a plurality of lysates from different hostmicroorganisms as the source of the enzymes.

Enzymes Generating the C7 Aliphatic Backbone for Conversion to C6Building Blocks

In some embodiments, (S)-2-amino-6-oxopimelate in FIG. 1 is substitutedwith the central precursor N-Acetyl-L-2-amino-6-oxopimelate.

In some embodiments, (S)-2-amino-6-oxopimelate in FIG. 1 is substitutedwith the central precursor N-Succinyl-2-L-amino-6-oxoheptanedioate.

In some embodiments, the C7 aliphatic backbone can be enzymaticallyformed from meso-2,6-diaminopimelate using one or more of adehydrogenase, a CoA-transferase, a dehydratase, a reductase, a mutase,a CoA-ligase, an ammonia lyase and a thioesterase. See, FIGS. 1 and 2.

In some embodiments, the dehydrogenase is a diaminopimelatedehydrogenase classified, for example, under EC 1.4.1.16.

In some embodiments, the dehydrogenase is a (R)-2-hydroxyisocaproatedehydrogenase such as the gene product of LdhA or a 2-hydroxyglutaratedehydrogenase such as the gene product of HgdH.

In some embodiments, the CoA-transferase is a glutaconateCoA-transferase, classified, for example, under EC 2.8.3.12, such as thegene product of GetAB or a pimelate CoA-transferase classified, forexample, under EC 2.8.3.—such as the gene product of thnH.

In some embodiments, the CoA-ligase is a succinate CoA-ligase, forexample, under EC 6.2.1.5.

In some embodiments, the dehydratase is a 2-hydroxyisocaproyl-CoAdehydratase such as SEQ ID NO: 28 or a 2-hydroxyglutaryl-CoA dehydratasesuch as SEQ ID NO: 25.

In some embodiments, the thioesterase is classified, for example, underEC 3.1.2.-, such as that encoded by YciA, tesB, acot13, SEQ ID NO: 1 orSEQ ID NO: 2.

In some embodiments, the reductase is a carboxylate reductaseclassified, for example, under EC 1.2.99.6 such as the gene products ofcar & npt, GriC & GriD or SEQ ID NO: 5, 7.

In some embodiments, the reductase is an enoate reductase (old yellowenzyme) classified, for example, under EC 1.3.1.31 or EC 1.6.99.1 suchas the gene product of SEQ ID NO: 16-22.

In some embodiments, the dehydrogenase is an aldehyde dehydrogenaseclassified, for example, under EC 1.2.1.—such as EC 1.2.1.3.

In some embodiments, the mutase is a lysine 2,3-aminomutase classified,for example, under EC 5.4.3.2 such as SEQ ID NO: 26.

In some embodiments, the ammonia lyase is a 3-butyryl-CoA ammonia lyaseclassified, for example, under EC 4.3.1.14 such as SEQ ID NO: 23.

Enzymes Generating the Terminal Carboxyl Groups in the Biosynthesis ofC6 Building Blocks

As depicted in FIG. 1, FIG. 2, and FIG. 3, a terminal carboxyl group canbe enzymatically formed using an aldehyde dehydrogenase, a thioesterase,a CoA-transferase, or a CoA-ligase.

In some embodiments, the first terminal carboxyl group leading to thesynthesis of adipic acid is enzymatically formed by an aldehydedehydrogenase classified, for example, under EC 1.2.1.3 (Guerrillot &Vandecasteele, Eur. J. Biochem., 1977, 81, 185-192). See, e.g., FIG. 3.

In some embodiments, the second terminal carboxyl group leading to thesynthesis of a C6 building block is enzymatically formed by an acyl-CoAhydrolase or thioesterase classified under EC 3.1.2.-, such as the geneproduct of YciA, tesB, Acot13, SEQ ID NO: 1 or SEQ ID NO: 2 (see, forexample, Cantu et al., Protein Science, 2010, 19, 1281-1295; Zhuang etal., Biochemistry, 2008, 47(9), 2789-2796; or Naggert et al., Journal ofBiological Chemistry, 1991, 266(17), 11044-11050, Jing et al., BMCBiochemistry, 2011, 12, 44), See, e.g., FIG. 3.

In some embodiments, the second terminal carboxyl group leading to thesynthesis of a C6 building block is enzymatically formed by aCoA-transferase such as a glutaeonate CoA-transferase classified, forexample, under EC 2.8.3.12. See, e.g., FIG. 3.

In some embodiments, the second terminal carboxyl group leading to thesynthesis of a C6 building block is enzymatically formed by a reversibleCoA-ligase such as succinate CoA-transferase classified under EC6.2.1.5. See, e.g., FIG. 3.

In some embodiments, the second terminal carboxyl group leading to thesynthesis of adipic acid is enzymatically formed by an aldehydedehydrogenase classified, for example, under EC 1.2.1.63, such as thegene product of ChnE (Iwaki et al., Appl. Environ. Microbial., 1999,65(11), 5158-5162). See, FIG. 3.

Enzymes Generating the Terminal Amine Groups in the Biosynthesis of C6Building Blocks

As depicted in FIG. 5, FIG. 6, and FIG. 7 a terminal amine group can beenzymatically formed using a ω-transaminase.

In some embodiments, a terminal amine group is enzymatically formed by aω-transaminase classified, for example, under EC 2.6.1.-, e.g., EC2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82 such asthat obtained from Chromobacterium violaceum (Genbank Accession No.AAQ59697.1), Pseudomonas aeruginosa (Genbank Accession No. AAG08191.1),Pseudomonas syringae (Genbank Accession No. AAY39893.1), Rhodobactersphaeroides (Genbank Accession No, ABA81135.1), Vibrio fluvialis(Genbank Accession No. AEA39183.1), Streptomyces griseus, or Clostridiumviride. See, FIG. 3.

An additional ω-transaminase that can be used in the methods and hostsdescribed herein is from Escherichia coli (Genbank Accession No.AAA57874.1). Some of the ω-transaminases classified, for example, underEC 2.6.1.29 or EC 2.6.1.82 are diamine ω-transaminases.

In some embodiments, the first terminal amine group leading to thesynthesis of 6-aminohexanoic acid is enzymatically formed by aω-transaminase classified under EC 2.6.1.18, such as that obtained fromVibrio fluvialis or Chromobacterium violaceum, EC 2.6.1.19, such as thatobtained from Streptomyces griseus, or EC 2.6.1.48, such as thatobtained from Clostridium viride.

The reversible ω-transaminase from Chromobacterium violaceum hasdemonstrated analogous activity accepting 6-aminohexanoic acid as aminodonor, thus forming the first terminal amine group in adipateseminaldehyde (Kaulmann et al., Enzyme and Microbial Technology, 2007,41, 628-637).

The reversible 4-aminobutyryl: 2-oxoglutarate transaminase fromStreptomyces griseus has demonstrated analogous activity for theconversion of 6-aminohexanoic acid to adipate semialdehyde (Yonaha etal., Eur. J. Biochem., 1985, 146, 101 - 106).

The reversible 5-aminovalerate transaminase from Clostridium viride hasdemonstrated analogous activity for the conversion of 6-aminohexanoicacid to adipate semialdehyde (Barker et al., The Journal of BiologicalChemistry, 1987, 262(19), 8994-9003).

In some embodiments, the second terminal amine group leading to thesynthesis of hexamethylenediamine is enzymatically formed by atransaminase classified under EC 2.6.1.29 or classified under EC2.6.1.82, such as the gene product of YgjG.

The gene product of ygjG accepts a broad range of diamine carbon chainlength substrates, such as putrescine, cadaverine and spermidine(Samsonova et al., BMC Microbiology, 2003, 3:2).

The diamine transaminase from E. coli strain B has demonstrated activityfor 1,6 diaminohexane (Kim, The Journal of Chemistry, 1963, 239(3),783-786)

Enzymes Generating the Terminal Hydroxyl Groups in the Biosynthesis ofC6 Building Blocks

As depicted in FIG. 8, the terminal hydroxyl group can be enzymaticallyforming using an alcohol dehydrogenase.

In some embodiments, the first terminal hydroxyl group leading to thesynthesis of 1,6 hexanediol is enzymatically formed by an alcoholdehydrogenase classified, for example, under EC 1,1.1.2 such as the geneproduct of YMR318C or an alcohol dehydrogenase classified, for example,under EC 1.1.1.258 such as the gene product of ChnI).

In some embodiments, the second terminal hydroxyl group leading to thesynthesis of 1,6 hexanediol is enzymatically formed by an alcoholdehydrogenase classified under EC 1.1.1.- (e.g., 1, 2, 21, or 184).

Biochemical Pathways Pathways to (S) 2-Aminopimelate and (R)2-Aminopimelate as Precursor Leading to Central Precursors to C6Building Blocks

In some embodiments, (S) 2-aminopimelate is synthesized from the centralmetabolite, meso-2,6-diaminopimelate, by conversion ofmeso-2,6-diaminopimelate to (S)-2-amino-6-oxopimelate by adiaminopimelate dehydrogenase (classified for example under EC1.4.1.16); followed by conversion of (S)-2-amino-6-oxopimelate to (S,R)2-amino-6-hydroxypimelate by a (R)-2-hydroxylsocaproate dehydrogenase(classified for example under EC 1.1.1.337) such as the gene product ofLdhA or a (R) 2-hydroxyglutarate dehydrogenase such as the gene productof HgdH; followed by conversion of (S,R) 2-amino-6-hydroxypimelate to(R,S) 2-hydroxy-6-aminopimeloyl-CoA by a glutaconate CoA-transferase(classified, for example, under EC 2.8.3.12) such as the gene product ofGctAB; followed by conversion of (R,S) 2-hydroxy-6-aminopimeloyl-CoA to(S) 6-amino-2,3-dehydropimeloyl-CoA by a 2-hydroxylsocaproyl-CoAdehydratase such as SEQ ID NO: 28 activated SEQ ID NO: 27 or(R)-2-hydroxyglutryl-CoA dehydratase such as SEQ ID NO: 25 activated bySEQ ID NO: 24; followed by conversion of (S)6-amino-2,3-dehydropimeloyl-CoA to (S) 6-amino-2,3-dehydropimelate by aglutaconate CoA-transferase (classified, for example, under EC2.8.3.12); followed by conversion to (S) 2-amino-7-oxohept-6-enoate by acarboxylate reductase classified, for example, under EC 1.2.99.6) suchas the gene product of car & npt, GriC & GriD or a carboxylate reductasesuch as SEQ ID NO: 5, 7; followed by conversion to (S)2-amino-7-oxoheptanoate by an enoate reductase (classified, for example,under EC 1.3.1.31 or EC 1.6.99.1) such as the gene product of SEQ ID NO:16-22; followed by conversion to (S) 2-aminopimelate by an aldehydedehydrogenase (classified, for example, under EC 1.2.1.3). See FIG. 1.

In some embodiments, (S)-2-amino-6-oxopimelate in FIG. 1 is substitutedwith the central precursor N-Acetyl-L-2-amino-6-oxopimelate.

In some embodiments, (S)-2-amino-6-oxopimelate in FIG. 1 is substitutedwith the central precursor N-Succinyl-2-L-amino-6-oxoheptanedioate.

In some embodiments, (R) 2-aminopimelate is synthesized from the centralmetabolite, meso-2,6-diaminopimelate, by conversion ofmeso-2,6-diaminopimelate to (S,R) 3,6 diaminopimelate by a lysine2,3-aminomutase (classified, for example, under EC 5.4.3.2) such SEQ IDNO: 26; followed by conversion of (S,R) 3,6 diaminopimelate to (S,R) 3,6diaminopimeloyl-CoA by a succinate-CoA ligase (classified, for example,under EC 6.2.1.5); followed by conversion of (S,R) 3,6diaminopimeloyl-CoA to (R) 6-amino-2,3-dehydropimelloyl-CoA by a3-aminobutyl-CoA ammonia lyase (classified, for example, under EC4.3.1.14) such as SEQ ID NO: 23; followed by the conversion of (R)6-amino-2,3-dehydropimeloyl-CoA to (R) 6-amino-2,3-dehydropimelate by athioesterase (classified, for example, under EC 3.1.2.-) such as SEQ IDNO: 1-2 or the gene product of YciA, tesB or acot13 or by aCoA-transferase (classified, for example, under EC 2.8.3.-) such as thegene product of thnH; followed by conversion to (R)2-amino-7-oxohept-6-enoate by a carboxylate reductase (classified, forexample, under EC 1.2.99.6) such as the gene product of car & npt, GriC& GriD or the carboxylate reductase SEQ ID NO: 5,7; followed byconversion to (R) 2-amino-7-oxoheptanoate by an enoate reductase(classified, for example, under EC 1.3.1.31) such as SEQ ID NO: 16-22;followed by conversion to (R) 2-aminopimelate by an aldehydedehydrogenase (classified, for example, under EC 1.2.1.3). See FIG. 2.

Pathways using (S) 2-Aminopimelate or (R) 2-Aminopimelate as CentralPrecursor to Adipic Acid.

In some embodiments, adipic acid is synthesized from the centralprecursor (S) aminopimelate or (R) 2-aminopimelate by conversion of (S)2-aminopimelate to 2-oxopimelate by an L-specific alpha-aminotransferase(classified under EC 2.6.1.—such as EC 2.6.1.39 or EC 2.6.1.42) such asthe gene product of ilvE or by conversion of (R) 2-aminopimelate to2-oxopimelate by a D-specific alpha-aminotransferase (classified underEC 2.6.1,—such as EC 2.6.1.21) such as the gene product of D-AAAT;followed by conversion of 2-oxopimelate to adipate semialdehyde by abranch-chain-2-oxoacid decarboxylase (classified, for example, under EC4.1.1.—such as EC 4.1.1.43, EC 4.1.1.71, EC 4.1.1.72 or EC 4.1.1.74)such as SEQ ID NO: 34 or the gene product of kivD or kdca or anacetolactate synthase (classified, for example, under EC 2.2.1.6) suchas the gene product of ilvB & ilvN; followed by conversion of adipatesemialdehyde to adipic acid by an aldehyde dehydrogenase (classified,for example, under EC 1.2.1.—such as EC 1.2.1.3, EC 1.2.1.16, EC1.2.1.20, EC 1.2.1.63, EC 1.2.1.79) such as the gene product of ChnE,CpnE or ThnG. See FIG. 3.

In some embodiments, 2-oxopimelate obtained as described above isconverted to adipyl-CoA by a dehydrogenase complex (classified, forexample, under EC 1.2.4.2, EC 1.8.1.4, and EC 2.3.1.61); followed byconversion to adipic acid by a thioesterase (classified, for example,under EC 3.1.2,-) such as SEQ ID NO: 1-2 or the gene product of YciA,tesB or acot13 or by a glutaconate CoA-transferase (classified under,for example, EC 2.8.3.12) or a reversible succinate CoA-ligase(classified, for example, under EC 6.2.1.5). See FIG. 3.

Pathway using (R) 2-Aminopimelate or (S) 2-Aminopimelate as CentralPrecursor to 6-Aminohexanoate and ε-Caprolactam

In some embodiments, 6-aminohexanoic acid is synthesized from thecentral precursor (S) 2-aminopimelate, by conversion of (S)2-aminopimelate to 6-aminohexanoic acid by a decarboxylase (classified,for example, under EC 4.1.1.—such as EC 4.1.1.15, EC 4.1.1.17, EC4.1.1.18 or EC 4.1.1.19) such as SEQ ID NO: 29-32. See FIG. 4.

In some embodiments, 6-aminohexanoic acid is synthesized from thecentral precursor (R) 2-aminopimelate by conversion of (R)2-aminopimelate to 6-aminohexanoic acid by a decarboxylase (classified,for example, under EC 4.1,1.—such as EC 4.1.1.20) such as SEQ ID NO: 33.See FIG. 4.

In some embodiments, c-caprolactam is synthesized from the centralprecursor hexanoic acid by conversion of 6-aminohexanoic acid toε-caprolactam by a hydrolase (classified, for example, under EC3.5.2,-). See FIG. 4.

In some embodiments, 6-aminohexanoic acid is synthesized from thecentral precursor (S) 2-aminopimelate or (R) 2-aminopimelate byconversion of (S) 2-aminopimelate to 2-oxopimelate by an L-specificalpha-aminotransferase (classified under EC 2.6.1.—such as EC 2.6.1.39or EC 2.6.1.42) such as the gene product of ilvE or by conversion of (R)2-aminopimelate to 2-oxopimelate by a D-specific alpha-aminotransferase(classified under EC 2.6.1.—such as EC 2.6.1.21) such as the geneproduct of D-AAAT; followed by conversion of 2-oxopimelate to adipatesemialdehyde by a branch-chain-2-oxoacid decarboxylase (classified, forexample, under EC 4.1.1.—such as EC 4.1.1.43, EC 4.1.1.71, EC 4.1.1.72or EC 4.1.1.74) such as SEQ ID NO: 34 or the gene product of klvD orkdca or an acetolactate synthase (classified, for example, under EC2.2.1.6) such as the gene product of ilvB & ilvN; followed by conversionof adipate semialdehyde to 6-aminohexanoic acid by an ω-transaminase(classified, for example, under EC 2.6.1—such as EC 2.6.1.18, EC2.6.1.19, or EC 2.6.1.48, EC 2.6.1.29, or EC 2.6.1.82) such as SEQ ID NO8-13. See FIGS. 1, 2 and 4.

Pathway using 6-Aminohexanoic Acid as Central Precursor toHexamethylenediamine

In some embodiments, hexamethylenediamine is synthesized from thecentral precursor, 6-aminohexanoic acid, by conversion of6-aminohexanoic acid to 6-aminohexanal by a carboxylate reductase(classified under, for example, EC 1.2.99.6) such as the gene product ofcar alongside the gene product of npt or the gene product of GriC & GriD(Suzuki et al., J. Antibiot., 2007, 60(6), 380 387); followed byconversion of 6-aminohexanal to hexamethylenediamine by a ω-transaminase(classified, for example, under EC 2.6.1.18, EC 2.6.1.19, 2.6.1.48, EC2.6.1.29, or EC 2.6.1.82) such as SEQ ID NO: 8-13. See FIG. 5.

The carboxylate reductase encoded by the gene product of car andenhancer npt has broad substrate specificity, including terminaldifunctional C4 and C5 carboxylic acids (Venkitasubramanian et al.,Enzyme and Microbial Technology, 2008, 42, 130-137).

In some embodiments, 6-aminohexanoic acid is synthesized from thecentral precursor (S) 2-aminopimelate or (R) 2-aminopimelate byconversion of (S) 2-aminopimelate to 2-oxopimelate by an L-specificalpha-aminotransferase (classified under EC 2.6.1.—such as EC 2.6.1.39or EC 2.6.1.42) such as the gene product of ilvE or by conversion of (R)2-aminopimelate to 2-oxopimelate by a D-specific alpha-aminotransferase(classified under EC 2.6.1.—such as EC 2.6.1.21) such as the geneproduct of D-AAAT; followed by conversion of 2-oxopimelate to adipatesemialdehyde by a branch-chain-2-oxoacid decarboxylase (classified, forexample, under EC 4.1.1.—such as EC 4.1.1.43, EC 4.1.1.71, EC 4.1.1.72or EC 4.1.1.74) such as SEQ ID NO: 34 or the gene product of kivD orkdca or an acetolactate synthase (classified, for example, under EC2.2.1.6) such as the gene product of ilvB & ilvN; followed by conversionof adipate semialdehyde to 1,6 hexanedial by a carboxylate reductase(classified, for example, under EC 1.2.99.6) such as SEQ ID NO: 7;followed by conversion of 1,6-hexanedial to 6-aminohexanal by anω-transaminase (classified, for example, under EC 2.6.1.—such as EC2.6.1.18, EC 2.6.1.19, or EC 2.6.1.48, EC 2.6.1.29, or EC 2.6.1.82);followed by conversion of 6-aminohexanal to hexamethylenediamine by aω-transaminase (classified, for example, under EC 2.6.1.—such as EC2.6.1.18, EC 2.6.1.19, or EC 2.6.1.48, EC 2.6.1.29, or EC 2.6.1.82) suchas SEQ ID NO: 8-13. See FIG. 1, 2 and 5.

In some embodiments, hexamethylenediamine is synthesized from thecentral precursor, 6-aminohexanoic acid, by conversion of6-aminohexanoic acid to N6-acetyl-6-aminohexanoic acid by aN-acetyltransferase classified, for example, under EC 2.3.1.32; followedby conversion of N6-acetyl-6-aminohexanoic acid toN6-acetyl-6-aminohexanal by a carboxylate reductase classified, forexample, under EC 1.2.99.6 such as SEQ ID NO: 5-7 or the gene product ofGriC & GriD (Suzuki et al., J. Antibiot., 2007, 60(6), 380-387);followed by conversion of N6-acetyl-6-aminohexanal toN6-acetyl-1,6-diaminohexane by a ω-transaminase (classified, forexample, under EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48 or EC2.6.1.82) such as SEQ ID NO: 8-13; followed by conversion ofN6-acetyl-1,6-diaminohexane to hexamethylenediamine by a deacetylase(classified, for example, under EC 3.5.1.17). See FIG. 6.

Pathway using 6-Hydroxyhexanoic Acid as Central Precursor toHexamethylenediamine

In some embodiments, hexamethylenediamine is synthesized from thecentral precursor, 6-hydroxyhexanoic acid, by conversion of6-hydroxyhexanoic acid to 6-hydroxyhexanal by a carboxylate reductase(classified, for example, under EC 1.2.99.6) such as SEQ ID NO: 3-7 orthe gene product of car alongside the gene product of npt or the geneproduct of GriC & GriD (Suzuki et al., J. Antibiot., 2007, 60(6), 380387); followed by conversion of 6-hydroxyhexanal to1-amino-6-hydroxy-hexane by a transaminase (classified, for example,under EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48 or EC 2.6.1.82)such as SEQ ID NO: 8-13; followed by conversion of1-amino-6-hydroxy-hexane to 6-aminohexanal by an alcohol dehydrogenaseclassified, for example, under EC 1.1.1.1 encoded by YMR318C, YqhD orthe protein having GenBank Accession No. CAA81612.1 (from Geobacillusstearothermophilus); followed by conversion of 6-aminohexanal tohexamethylenediamine by a ω-transaminase (classified, for example, underEC 2.6.1.18, EC 2.6.1.19, 2.6.1.48, EC 2.6.1.29, or EC 2.6.1.82.) suchas SEQ ID NO: 8-13. See FIG. 7.

Pathways using (R) 2-Aminopimelate or (S) 2-Aminopimelate as CentralPrecursor to 1,6-Hexanediol

In some embodiments, adipic acid is synthesized from the centralprecursor (S) 2-aminopimelate or (R) 2-aminopimelate by conversion of(S) 2-aminopimelate to 2-oxopimelate by an L-specificalpha-aminotransferase (classified under EC 2.6.1.—such as EC 2.6.1.39or EC 2.6.1.42) such as the gene product of ilvE or by conversion of (R)2-aminopimelate to 2-oxopimelate by D-specific alpha-aminotransferase(classified under EC 2.6.1.—such as EC 2.6.1.21) such as the geneproduct of D-AAAT; followed by conversion of 2-oxopimelate to adipatesemialdehyde by a branch-chain-2-oxoacid decarboxylase (classified, forexample, under EC 4.1.1.—such as EC 4.1.1.43, EC 4.1.1.71, EC 4.1.1.72or EC 4.1.1.74) such as SEQ ID NO: 34 or the gene product of kivD orkdca or an acetolactate synthase (classified, for example, under EC2.2.1.6) such as the gene product of ilvB & ilbN; followed by conversionof adipate semialdehyde to 6-hydroxyhexanoic acid by an alcoholdehydrogenase (classified, for example, under EC 1.1.1.—such as EC1.1.1.2 or EC 1.1.1.258) such as encoded by YMR318C, ChnD, or gabD. See,FIG. 8.

In some embodiments, 1,6 hexanediol is synthesized from the centralprecursor 6-hydroxyhexanoic acid by conversion of 6-hydroxyhexanoic acidto 6-hydroxyhexanal by a carboxylate reductase (classified, for example,under EC 1.2.99.6) such as SEQ ID NO: 3-7; followed by conversion of6-hydroxyhexanal to 1,6 hexanediol by an alcohol dehydrogenase(classified, for example, under EC 1.1.1.- such as EC 1.1.1.1, :EC1.1.1.2, EC 1.1.1.21, or EC 1.1.1.184) such as encoded by YMR318C, YqhDor CAA81612.1 (Liu et al., Microbiology, 2009, 155, 2078-2085).

Cultivation Strategy

In some embodiments, one or more C6 building blocks are biosynthesizedin a recombinant host using anaerobic, aerobic or micro-aerobiccultivation conditions. In some embodiments, the cultivation strategyentails nutrient limitation such as nitrogen, phosphate or oxygenlimitation.

In some embodiments in which (S) 2-aminopimelate is produced as acentral precursor, a cultivation strategy entails either achieving ananaerobic or micro-aerobic cultivation condition.

In some embodiments in which (R) 2-aminopimelate is produced as acentral precursor, a cultivation strategy entails either achieving ananaerobic, aerobic or micro-aerobic cultivation condition.

In some embodiments, a cell retention strategy using, for example,ceramic hollow fiber membranes is employed to achieve and maintain ahigh cell density during either fed-batch or continuous fermentation.

In some embodiments, the cultivation strategy entails culturing underconditions of nutrient limitation either via nitrogen, phosphate oroxygen limitation.

In some embodiments, the principal carbon source fed to the fermentationin the synthesis of one or more C6 building blocks can derive frombiological or non-biological feed stocks.

In some embodiments, the biological feedstock can be or can derive from,monosaccharides, disaccharides, lignocellulose, hemicellulose,cellulose, lignin, levulinic acid and formic acid, triglycerides,glycerol, fatty acids, agricultural waste, condensed distillers'solubles, or municipal waste.

The efficient catabolism of crude glycerol stemming from the productionof biodiesel has been demonstrated in several microorganisms such asEscherichia coli, Cupriavidus necator, Pseudomonas oleavorans,Pseudomonas putida and Yarrowia lipolytica (Lee et al., Appl. Biochem.Biotechnol., 2012, 166:1801-1813; Yang et al., Biotechnology forBiofuels, 2012, 5:13; Meijnen et al. Appl. Microhiol. Biotechnol., 2011,90:885 893).

The efficient catabolism of lignocellulosic-derived levulinic acid hasbeen o demonstrated in several organisms such as Cupriavidus necator andPseudomonas putida in the synthesis of 3-hydroxyvalerate via theprecursor propanoyl-CoA (Jaremko and Yu, 2011, supra; Martin andPrather, J. Biotechnol., 2009, 139:61 67).

The efficient catabolism of lignin-derived aromatic compounds such asbenzoate analogues has been demonstrated in several microorganisms suchas Pseudomonas putida, Cupriavidus necator (Bugg et al., Current Opinionin Biotechnology, 2011, 22, 394-400; Pérez-Pantoja et al., FEMSMicrobiol. Rev., 2008, 32, 736-794).

The efficient utilization of agricultural waste, such as olive millwaste water has been demonstrated in several microorganisms, includingYarrowia lipolytica (Papanikolaou et al., Bioresour. Technol., 2008,99(7):2419-2428).

The efficient utilization of fermentable sugars such as monosaccharidesand disaccharides derived from cellulosic, hemicellulosic, cane and beetmolasses, cassava, corn and other agricultural sources has beendemonstrated for several microorganism such as Escherichia coli,Corynebacterium glutamicum and Lactobacillus delbrueckii and Lactococcuslactis (see, e.g., Hermann et al, J. Biotechnol., 2003, 104:155-172; Weeet al., Food Technol. Biotechnol., 2006, 44(2):163-172; Ohashi et al.,J. Bioscience and Bioengineering, 1999, 87(5):647-654).

The efficient utilization of furfural, derived from a variety ofagricultural lignocellulosic sources, has been demonstrated forCupriavidus necator (Li et al., Biodegradation, 2011, 22:1215-1225).

In some embodiments, the non-biological feedstock can be or can derivefrom natural gas, syngas, CO₂/H₂, methanol, ethanol, benzoate,non-volatile residue (NVR) or a caustic wash waste stream fromcyclohexane oxidation processes, or terephthalic acid/isophthalic acidmixture waste streams.

The efficient catabolism of methanol has been demonstrated for themethylotrophic yeast Pichia pastoris.

The efficient catabolism of ethanol has been demonstrated forClostridium kluyveri (Seedorf et al., Proc. Natl. Acad. Sci. USA, 2008,105(6) 2128-2133).

The efficient catabolism of CO₂ and H₂, which may be derived fromnatural gas and other chemical and petrochemical sources, has beendemonstrated for Cupriavidus necator (Prybylski et al., Energy,Sustainability and Society, 2012, 2:11).

The efficient catabolism of syngas has been demonstrated for numerousmicroorganisms, such as Clostridium ljungdahlii and Clostridiumautoethanogenum (Kopke et al., Applied and Environmental Microbiology,2011, 77(15):5467-5475).

The efficient catabolism of the non-volatile residue waste stream fromcyclohexane processes has been demonstrated for numerous microorganisms,such as Delftia acidovorans and Cupriavidus necator (Ramsay et al.,Applied and Environmental Microbiology, 1986, 52(1):152-156).

In some embodiments, the host microorganism is a prokaryote. Forexample, the prokaryote can be a bacterium from the genus Escherichiasuch as Escherichia coli; from the genus Clostridia such as Clostridiumljungdahlii, Clostridium autoethanogenum or Clostridium ktuyveri; fromthe genus Corynebacteria such as Corynebacterium glutamicum; from thegenus Cupriavidus such as Cupriavidus necator or Cupriavidusmetallidurans; from the genus Pseudomonas such as Pseudomonasfluorescens, Pseudomonas putida or Pseudomonas oleavorans; from thegenus Delftia such as Delftia acidovorans; from the genus Bacillus suchas Bacillus subtillis; from the genus Lactobacillus such asLactobacillus delbrueckii; or from the genus Lactococcus such asLactococcus lactis. Such prokaryotes also can be a source of genes toconstruct recombinant host cells described herein that are capable ofproducing one or more C6 building blocks.

In some embodiments, the host microorganism is a eukaryote. For example,the eukaryote can be a filamentous fungus, e.g., one from the genusAspergillus such as Aspergillus niger. Alternatively, the eukaryote canbe a yeast, e.g., one from the genus Saccharomyces such as Saccharomycescerevisiae; from the genus Pichia such as Pichia pastoris; or from thegenus Yarrowia such as Yarrowia lipolytica; from the genus Issaichenkiasuch as Issathenkia orientalis; from the genus Debaryomyces such asDebaryomyces hansenii; from the genus Arxula such as Arxsulaadenoinivorans; or from the genus Kluyveromyces such as Kluyveromyceslactis. Such eukaryotes also can be a source of genes to constructrecombinant host cells described herein that are capable of o producingone or more C6 building blocks.

Metabolic Engineering

The present document provides methods involving less than all the stepsdescribed for all the above pathways. Such methods can involve, forexample, one, two, three, four, five, six, seven, eight, nine, ten, ormore of such steps. Where less than all the steps are included in such amethod, the first step can be any one of the steps listed.

Furthermore, recombinant hosts described herein can include anycombination of the above enzymes such that one or more of the steps,e.g., one, two, three, four, five, six, seven, eight, nine, ten, or moreof such steps, can be performed within a recombinant host. This documentprovides host cells of any of the genera and species listed andgenetically engineered to express one or more (e.g., two, three, four,five, six, seven, eight, nine, 10, 11, 12 or more) recombinant forms ofany of the enzymes recited in the document. Thus, for example, the hostcells can contain exogenous nucleic acids encoding enzymes catalyzingone or more of the steps of any of the pathways described herein.

In addition, this document recognizes that where enzymes have beendescribed as accepting CoA-activated substrates, analogous enzymeactivities associated with [acp]-bound substrates exist that are notnecessarily in the same enzyme class.

Also, this document recognizes that where enzymes have been describedaccepting (R)-enantiomers of substrate, analogous enzyme activitiesassociated with (S)-enantiomer substrates exist that are not necessarilyin the same enzyme class.

This document also recognizes that where an enzyme is shown to accept aparticular co-factor, such as NADPH, or co-substrate, such asacetyl-CoA, many enzymes are promiscuous in terms of accepting a numberof different co-factors or co-substrates in catalyzing a particularenzyme activity. Also, this document recognizes that where enzymes havehigh specificity for e.g., a particular co-factor such as NADH, anenzyme with similar or identical activity that has high specificity forthe co-factor NADPH may be in a different enzyme class.

In some embodiments, the enzymes in the pathways outlined in section 4.5are the result of enzyme engineering via non-direct or rational enzymedesign approaches with aims of improving activity, improvingspecificity, reducing feedback inhibition, reducing repression,improving enzyme solubility, changing stereo-specificity, or changingco-factor specificity.

In some embodiments, the enzymes in the pathways outlined in section 4.5are gene dosed (i.e., overexpressed by having a plurality of copies ofthe gene in the host organism), into the resulting genetically modifiedorganism via episomal or chromosomal integration approaches.

In some embodiments, genome-scale system biology techniques such as FluxBalance Analysis are utilized to devise genome scale attenuation orknockout strategies for directing carbon flux to a C6 building block.

Attenuation strategies include, but are not limited to; the use oftransposons, homologous recombination (double cross-over approach),mutagenesis, enzyme inhibitors and RNA interference (RNAi).

In some embodiments, fluxomic, metabolomic and transcriptomal data areutilized to inform or support genome-scale system biology techniques,thereby devising genome scale attenuation or knockout strategies indirecting carbon flux to a C6 building block.

In some embodiments, the host microorganism's tolerance to highconcentrations of a C6 building block is improved through continuouscultivation in a selective environment.

In some embodiments, the host microorganism's biochemical network isattenuated or augmented to (1) ensure the intracellular availability ofoxaloacetate, (2) create an NADPH imbalance that may only be balancedvia the formation of one or more C6 building blocks, (3) preventdegradation of central metabolites, central precursors leading to andincluding C6 building blocks and (4) ensure efficient efflux from thecell.

In some embodiments, the anaplerotic reactions from glycolysis leadinginto the Krebs cycle to augment oxaloacetate are overexpressed in thehost.

In some embodiments where the host microorganism uses the lysinebiosynthesis pathway via meso-2,6-diaminopimelate, the genes encodingthe synthesis of lysine from 2-oxoglutarate via 2-oxoadipate are genedosed into the host organisms.

In some embodiments where the host microorganism uses the lysinebiosynthesis pathway via 2-oxoadipate, the genes encoding the synthesisof lysine via meso-2,6-diaminopimelate are gene dosed into the hostorganisms.

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of a C6 building block, a puridine nucleotidetranshydrogenase gene such as UdhA is overexpressed in the hostorganisms (Brigham et al., Advanced Biofuels and Bioproducts, 2012,Chapter 39, 1065-1090),

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of a C6 building block, a glyceraldehyde-3P-dehydrogenasegene such as GapN is overexpressed in the host organisms (Brigham etal., 2012, supra).

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of a C6 building block, a gene encoding a malic enzyme,such as mseA or maeB, is overexpressed in the host (Brigham et al.,2012, supra).

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of a C6 building block, a gene encoding aglucose-6-phosphate dehydrogenase such as overexpressed in the host (Limet al. Journal of Bioscience and Bioengineering, 2002, 93(6), 543-549).

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of a C6 building block, a gene encoding a fructose 1,6diphosphatase such as) fbp is overexpressed in the host (Becker et al.,Journal of Biotechnology, 2007, 132, 99-109).

In some embodiments, where pathways require excess NADPB co-factor inthe synthesis of a C5 building block, an endogenous gene encoding atriose phosphate isomerase (EC 5.3.1.1) is attenuated.

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of a C6 building block, an endogenous gene encoding aglucose dehydrogenase o such as the gene product of gdh is overexpressedin the host (Satoh et al., Journal of Bioscience and Bioengineering,2003, 95(4), 335-341).

In some embodiments, endogenous genes encoding enzymes facilitating theconversion of NADPH to NADH are attenuated, such as the NADH generationcycle that may be generated via inter-conversion of a glutamatedehydrogenase in EC 1.4.1.2 (NADH-specific) and EC 1.4.1.4(NADPH-specific).

In some embodiments, an endogenous gene encoding a glutamatedehydrogenase (EC 1.4.1.3) that can utilize both NADH and NADPH asco-factors is attenuated.

In some embodiments using hosts that naturally accumulatepolyhydroxyalkanoates, one or more endogenous genes encoding polymersynthase enzymes can be attenuated in the host strain.

In some embodiments, β-oxidation enzymes degrading central metabolitesand central precursors leading to and including C6 building blocks areattenuated.

In some embodiments, enzymes activating C6 building blocks via CoenzymeA esterification such as CoA-ligases are attenuated.

In some embodiments, the efflux of a C6 building block across the cellmembrane to the extracellular media is enhanced or amplified bygenetically engineering structural modifications to the cell membrane orincreasing any associated transporter activity for a C6 building block.

Producing C6 Building Blocks Using a Recombinant Host

Typically, one or more C6 building blocks can be produced by providing ahost microorganism and culturing the provided microorganism with aculture medium containing a suitable carbon source as described above.In general, the culture media and/or culture conditions can be such thatthe microorganisms grow to an adequate density and produce a C6 buildingblock efficiently. For large-scale production processes, any method canbe used such as those described elsewhere (Manual of IndustrialMicrobiology and Biotechnology, 2^(nd) Edition, Editors: A. L. Demainand J. E. Davies, ASM Press; and Principles of Fermentation Technology,P. F. Stanbury and A. Whitaker, Pergamon), Briefly, a large tank (e.g.,a 100 gallon, 200 gallon, 500 gallon, or more tank) containing anappropriate culture medium is inoculated with a particularmicroorganism. After inoculation, the microorganism is incubated toallow biomass to be produced. Once a desired biomass is reached, thebroth containing the microorganisms can be transferred to a second tank.This second tank can be any size. For example, the second tank can belarger, smaller, or the same size as the first tank. Typically, thesecond tank is larger than the first such that additional culture mediumcan be added to the broth from the first tank. In addition, the culturemedium within this second tank can be the same as, or different from,that used in the first tank.

Once transferred, the microorganisms can be incubated to allow for theproduction of a C6 building block. Once produced, any method can be usedto isolate C6 building blocks. For example, C6 building blocks can berecovered selectively from the fermentation broth via adsorptionprocesses. In the case of adipic acid and 6-aminoheptanoic acid, theresulting eluate can be further concentrated via evaporation,crystallized via evaporative and/or cooling crystallization, and thecrystals recovered via centrifugation. In the case ofhexamethylenediamine and 1,6-hexanediol, distillation may be employed toachieve the desired product purity.

EXAMPLES Example 1 Enzyme Activity of ω-Transaminase using AdipateSemialdehyde as Substrate and Forming 6-Aminohexanoate

A nucleotide sequence encoding a His-tag was added to the genes fromChromobacterium violaceum, Pseudomonas aeruginosa, Pseudomonas syringae,Rhodobacter sphaeroides, and Vibrio Fluvialis encoding theω-transaminases of SEQ ID NOs: 8, 9, 10, 11 and 13, respectively (seeFIG. 20E and FIG. 20F) such that N-terminal HIS tagged ω-transaminasescould be produced. Each of the resulting modified genes was cloned intoa pET21a expression vector under control of the T7 promoter and eachexpression vector was transformed into a BL21[DE3] E. coli host. Theresulting recombinant E. coli strains were cultivated at 37° C. in a 250mL shake flask culture containing 50 mL LB media and antibioticselection pressure, with shaking at 230 rpm. Each culture was inducedovernight at 16° C, using 1 mM IPTG.

The pellet from each induced shake flask culture was harvested viacentrifugation. Each pellet was resuspended and lysed via sonication.The cell debris was separated from the supernatant via centrifugationand the cell free extract was used immediately in enzyme activityassays.

Enzyme activity assays in the reverse direction (i.e., 6-aminohexanoateto adipate semialdehyde) were performed in a buffer composed of a finalconcentration of 50 mM HEPES buffer (pH=7.5), 10 mM 6-aminohexanoate, 10mM pyruvate and 100 μM pyridoxyl 5′ phosphate. Each enzyme activityassay reaction was initiated by adding cell free extract of theo-transaminase gene product or the empty vector control to the assaybuffer containing the 6-aminohexanoate and incubated at 25° C. for 24 h,with shaking at 250 rpm. The formation of L-alanine from pyruvate wasquantified via RP-HPLC.

Each enzyme only control without 6-aminohexanoate demonstrated low baseline conversion of pyruvate to L-alanine. See FIG. 14. The gene productof SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 11 and SEQ ID NO 13 accepted6-aminohexanote as substrate as confirmed against the empty vectorcontrol. See FIG. 15.

Enzyme activity in the forward direction (i.e., adipate semialdehyde to6-aminohexanoate) was confirmed for the transaminases of SEQ ID NO 8,SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11 and SEQ ID NO 13. Enzymeactivity assays were performed in a buffer composed of a finalconcentration of 50 mM HEPES buffer (pH=7.5), 10 mM. adipatesemialdehyde, 10 mM L-alanine and 100 μM pyridoxyl 5′ phosphate. Eachenzyme activity assay reaction was initiated by adding a cell freeextract of the co-transaminase gene product or the empty vector controlto the assay butler containing the adipate semialdehyde and incubated at25° C. for 4 h, with shaking at 250 rpm. The formation of pyruvate wasquantified via RP-HPLC.

The gene product of SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11and SEQ ID NO 13 accepted adipate semialdehyde as substrate as confirmedagainst the empty vector control. See FIG. 16. The reversibility of theω-transaminase activity was confirmed, demonstrating that theω-transaminases of SEQ NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11and. SEQ ID NO 13 accepted adipate semialdehyde as substrate andsynthesized 6-aminohexanoate as a reaction product.

Example 2 Enzyme Activity of Carboxylate Reductase using6-Hydroxyhexanoate as Substrate and Forming 6-Hydroxyhexanal

A nucleotide sequence encoding a His-tag was added to the genes fromMycobacterium marinum, Mycobacterium smegmatis, Mycobacterium smegmatis,Segniliparus rugosus, Mycobacterium massiliense, and Segniliparusrotundus that encode the carboxylate reductases of SEQ NOs: 3-7,respectively (see FIGS. 20A-20E) such that N-terminal HIS taggedcarboxylate reductases could be produced. Each of the modified genes wascloned into a pET Duet expression vector alongside a sfp gene encoding aHis-tagged phosphopantetheine transferase from Bacillus subtilis, bothunder control of the T7 promoter. Each expression vector was transformedinto a BL21[DE3]E. coli host. Each resulting recombinant E. coli strainwas cultivated at 37° C. in a 250 mL shake flask culture containing 50mL LB media and antibiotic selection pressure, with shaking at 230 rpm.Each culture was induced overnight at 37° C. using an auto-inductionmedia.

The pellet from each induced shake flask culture was harvested viacentrifugation. Each pellet was resuspended and lysed via sonication.The cell debris was separated from the supernatant via centrifugation.The carboxylate reductases and phosphopantetheine transferase werepurified from the supernatant using Ni-affinity chromatography, diluted10-fold into 50 mM HEPES buffer (pH=7.5) and concentrated viaultrafiltration.

Enzyme activity (i.e., 6-hydroxyhexanoate to 6-hydroxyhexanal) assayswere performed in triplicate in a buffer composed of a finalconcentration of 50 mM HEPES buffer (pH=7.5), 2 mM 6-hydroxyhexanal, 10mM MgCl₂, 1 mM ATP, and 1 mM NADPH. Each enzyme activity assay reactionwas initiated by adding purified carboxylate reductase andphosphopantetheine transferase or the empty vector control to the assaybuffer containing the 6-hydroxyhexanoate and then incubated at room otemperature for 20 min. The consumption of NADPH was monitored byabsorbance at 340 nm. Each enzyme only control without6-hydroxyhexanoate demonstrated low base line consumption of NADPH. SeeFIG. 9.

The gene products of SEQ ID NO 3-7, enhanced by the gene product of sfp,accepted 6-hydroxyhexanoate as substrate as confirmed against the emptyvector control (see FIG. 11), and synthesized 6-hydroxyhexanal.

Example 3 Enzyme Activity of ω-Transaminase for 6-Aminohexanol, Forming6-Oxoliexanol

A nucleotide sequence encoding an N-terminal His-tag was added to theChromobacterium violaceum, Pseudomonas aeruginosa, Pseudomonas syringae,Rhodobacter sphaeroides, Escherichia coli, and Vibrio fluvialis genesencoding the ω-transaminases of SEQ ID NOs: 8-13, respectively (see FIG.20E and FIG. 20F) such that N-terminal HIS tagged ω-transaminases couldbe produced. The modified genes were cloned into a pET21a expressionvector under the T7 promoter. Each expression vector was transformedinto a BL21.[DE3] E. coli host. Each resulting recombinant E. colistrain were cultivated at 37° C. in a 250 mL shake flask culturecontaining 50 mL LB media and antibiotic selection pressure, withshaking at 230 rpm. Each culture was induced overnight at 16° C. using 1mM IPTG.

The pellet from each induced shake flask culture was harvested viacentrifugation. Each pellet was resuspended and lysed via sonication.The cell debris was separated from the supernatant via centrifugationand the cell free extract was used immediately in enzyme activityassays.

Enzyme activity assays in the reverse direction (i.e., 6-aminohexanol to6-oxohexanol) were performed in a buffer composed of a finalconcentration of 50 mM HEPES buffer (pH=7.5), 10 mM 6-aminohexanol, 10mM pyruvate, and 100 nM pyridoxyl 5′ phosphate. Each enzyme activityassay reaction was initiated by adding cell free extract of theω-transaminase gene product or the empty vector control to the assaybuffer containing the 6-aminohexanol and then incubated at 25° C. for 4h, with shaking at 250 rpm. The formation of L-alanine was quantifiedvia RP-HPLC.

Each enzyme only control without 6-aminohexanol had low base lineconversion of pyruvate to L-alanine. See FIG. 14.

The gene products of SEQ ID NO 8-13 accepted 6-aminohexanol as substrateas confirmed against the empty vector control (see FIG. 19) andsynthesized 6-oxohexanol as reaction product. Given the reversibility ofthe ω-transaminase activity (see Example 1), it can be concluded thatthe gene products of SEQ ID 8-13 accept 6-aminohexanol as substrate andform 6-oxohexanol.

Example 4 Enzyme Activity of ω-Transaminase using Hexamethylenediamineas Substrate and Forming 6-Aminohexanal

A nucleotide sequence encoding an N-terminal His-tag was added to theChromobacterium violaceum, Pseudomonas aeruginosa, Pseudomonas syringae,Rhodobacter sphaeroides, Escherichia coli, and Vibrio fluvialis genesencoding the ω-transaminases of SEQ ID NOs: 8-13, respectively (see FIG.20E and FIG. 20F) such that N-terminal HIS tagged ω-transaminases couldbe produced. The modified genes were cloned into a pET21a expressionvector under the T7 promoter. Each expression vector was transformedinto a BL21.[DE3] E. coli host. Each resulting recombinant E. colistrain were cultivated at 37° C. in a 250 mL shake flask culturecontaining 50 mL LB media and antibiotic selection pressure, withshaking at 230 rpm. Each culture was induced overnight at 16° C. using 1mM IPTG.

The pellet from each induced shake flask culture was harvested viacentrifugation. Each pellet was resuspended and lysed via sonication.The cell debris was separated from the supernatant via centrifugationand the cell free extract was used immediately in enzyme activityassays.

Enzyme activity assays in the reverse direction (i.e.,hexamethylenediamine to 6-aminohexanal) were performed in a buffercomposed of a final concentration of 50 mM HEPES buffer (pH=7.5), 10 mMhexamethylenediamine, 10 mM pyruvate, and 100 μM pyridoxyl 5′ phosphate.Each enzyme activity assay reaction was initiated by adding cell freeextract of the ω-transaminase gene product or the empty vector controlto the assay buffer containing the hexamethylenediamine and thenincubated at 25° C. for 4 h, with shaking at 250 rpm. The formation ofL-alanine was quantified via RP-HPLC.

Each enzyme only control without hexamethylenediamine had low base lineconversion of pyruvate to L-alanine. See FIG. 14.

The gene products of SEQ ID NO 8-13 accepted hexamethylenediamine assubstrate as confirmed against the empty vector control (see FIG. 17)and synthesized 6-aminohexanal as reaction product. Given thereversibility of the ω-transaminase activity (see Example 1), it can beconcluded that the gene products of SEQ ID NOs: 8-13 accept6-aminohexanal as substrate and form hexamethylenediamine.

Example 5 Enzyme Activity of Carboxylate Reductase forN6-Acetyl-6-Aminohexanoate, Forming N6-Acetyl-6-Aminohexanal

The activity of each of the N-terminal His-tagged carboxylate reductasesof SEQ ID NOs: 5-7 (see Example 2, and FIGS. 20C-20E) for convertingN6-acetyl-6-aminohexanoate to N6-acetyl-6-aminohexanal was assayed intriplicate in a buffer composed of a final concentration of 50 mM HEPESbuffer (pH=7.5), 2 mM N6-acetyl-6-aminohexanoate, 10 mM MgCl₂, 1 mM ATP,and 1 mM NADPH. The assays were initiated by adding purified carboxylatereductase and phasphopantetheine transferase or the empty vector controlto the assay buffer containing the N6-acetyl-6-aminohexanoate thenincubated at room temperature for 20 min. The consumption of NADPH wasmonitored by absorbance at 340 nm. Each enzyme only control withoutN6-acetyl-6-aminohexanoate demonstrated low base line consumption ofNADPH. See FIG. 9.

The gene products of SEQ ID NO 5-7, enhanced by the gene product ofaccepted N6-acetyl-6-aminohexanoate as substrate as confirmed againstthe empty vector control (see FIG. 12), and synthesizedN6-acetyl-6-aminohexanal.

Example 6 Enzyme Activity of ω-Transaminase usingN6-Acetyl-1,6-Diaminohexane, and Forming N6-Acetyl-6-Aminohexanal

The activity of the N-terminal His-tagged ω-transaminases of SEQ ID NOs:8-13 (see Example 4, and FIG. 20E and FIG. 20F) for convertingN6-acetyl-1,6-diaminohexane to N6-acetyl-6-aminohexanal was assayedusing a buffer composed of a final concentration of 50 mM HEPES butler(pH=7.5), 10 mM. N6-acetyl-1,6-diaminohexane, 10 mM pyruvate and 100 μMpyridoxyl 5′ phosphate. Each enzyme activity assay reaction wasinitiated by adding a cell free extract of the co-transaminase or theempty vector control to the assay buffer containing theN6-acetyl-1,6-diaminohexane then incubated at 25° C. for 4 h, withshaking at 250 rpm. The formation of L-alanine was quantified viaRP-HPLC.

Each enzyme only control without N6-acetyl-1,6-diaminohexanedemonstrated low base line conversion of pyruvate to L-alanine. See FIG.14.

The gene product of SEQ ID NO 8-13 accepted N6-acetyl-1,6-diaminohexaneas substrate as confirmed against the empty vector control (see FIG. 18)and synthesized N6-acetyl-6-aminohexanal as reaction product.

Given the reversibility of the ω-transaminase activity (see example 1),the gene products of SEQ ID 8-13 accept N6-acetyl-6-aminohexanal assubstrate forming N6-acetyl-1,6-diaminohexane.

Example 7 Enzyme Activity of Carboxylate Reductase using AdipateSemialdehyde as Substrate and Forming Hexanedial

The N-terminal His-tagged carboxylate reductase of SEQ ID NO 7 (seeExample 2 and FIG. 20E) was assayed using adipate semialdehyde assubstrate. The enzyme activity assay was performed in triplicate in abuffer composed of a final concentration of 50 mM HEPES buffer (pH=7.5),2 mM adipate semialdehyde, 10 mM MgCl₂, 1 mM ATP and 1 mM NADPH. Theenzyme activity assay reaction was initiated by adding purifiedcarboxylate reductase and phosphopanteiheine transferase or the emptyvector control to the assay buffer containing the adipate semialdehydeand then incubated at room temperature for 20 min. The consumption ofNADPH was monitored by absorbance at 340 nm. The enzyme only controlwithout adipate semialdehyde o demonstrated low base line consumption ofNADPH. See FIG. 9.

The gene product of SEQ ID NO 7, enhanced by the gene product of sfp,accepted adipate semialdehyde as substrate as confirmed against theempty vector control (see FIG. 13) and synthesized hexanedial.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1-76. (canceled)
 77. A method of biosynthesizing at least one productchosen from adipic acid, adipate semialdehyde, 6-aminohexanoic acid,6-hydroxyhexanoic acid, caprolactam, hexamethylenediamine, and1,6-hexanediol in a recombinant host via fermentation, said methodcomprising: enzymatically converting 2,6-diaminopimelate to2-aminopimelate using at least one polypeptide having an activity chosenfrom 2-hydroxyacyl-CoA dehydratase activity, mutase activity, ammonialyase activity, and enoate reductase activity; and enzymaticallyconverting 2-aminopimelate to said at least one product.
 78. The methodof claim 77, wherein 2,6-diaminopimelate is enzymatically converted to(S) 2-aminopinelate or (R) 2-aminopimelate.
 79. The method of claim 77,said method comprising: using said polypeptide having 2-hydroxyacyl-CoAdehydratase activity and said polypeptide having enoate reductaseactivity to enzymatically convert 2,6-diaminopimelate to2-aminopimelate; and/or further using at least one polypeptide having anactivity chosen from diaminopimelate dehydrogenase activity,2-hydroxycarboxylate dehydrogenase activity, CoA-transferase activity,2-hydroxyacid dehydratase activity, and carboxylate reductase activityto enzymatically convert 2,6-diaminopimelate to 2-aminopimelate.
 80. Themethod of claim 77, said method comprising: using said polypeptidehaving mutase activity, said polypeptide having ammonia lyase activity,and said polypeptide having enoate reductase activity to enzymaticallyconvert 2,6-diaminopimelate to 2-aminopimelate; and/or further using atleast one polypeptide having an activity chosen from CoA ligaseactivity, CoA-transferase activity, carboxylate reductase activity, andaldehyde dehydrogenase activity to enzymatically convert2,6-diaminopimelate to 2-aminopimelate.
 81. The method of claim 77,wherein: said polypeptide having enoate reductase activity has at least70% sequence identity to the amino acid sequence set forth in any one ofSEQ ID NOs: 16-22; said polypeptide having 2-hydroxyacyl-CoA dehydrataseactivity has at least 70% sequence identity to the amino acid sequenceset forth in SEQ ID NO: 25 or SEQ ID NO: 28; said polypeptide havingmutase activity has at east 70% sequence identity to the amino acidsequence set forth in SEQ ID NO: 26; or said polypeptide having ammonialyase activity has at least 70% sequence identity to the amino acidsequence set forth in SEQ ID NO:
 23. 82. The method of claim 77, wherein2-aminopimelate is enzymatically converted to said at least one productusing: at least one polypeptide having an activity chosen from α-oxoaciddecarboxylase activity classified under EC 4.1.1.-, α-aminoaciddecarboxylase activity classified under EC 4.1.1.-, synthase activity,and activity of a dehydrogenase complex; and optionally one or morepolypeptides having an activity chosen from aldehyde dehydrogenaseactivity, alcohol dehydrogenase activity, CoA-transferase activity,carboxylate reductase activity, α-aminotransferase activity,thioesterase activity, hydrolase activity, ω-transaminase activity,N-acetyltransferase activity, and deacylase activity.
 83. The method ofclaim 82, wherein: said polypeptide having α-oxoacid decarboxylaseactivity is classified under EC 4.1.1.43, EC 4.1.1.71, EC 4.1.1.72, orEC 4.1.1.74; said polypeptide having α-aminoacid decarboxylase activityis classified under EC 4.1.1.15, EC 4.1.1.1 EC 4.1.1.18, or EC 4.1.1.19;said polypeptide having synthase activity is classified under EC2.2.1.6; or said polypeptide having the activity of a dehydrogenasecomplex comprises activities classified under EC 1.2.4.2, EC 1.8.1.4, orEC 2.3.1.61.
 84. The method of claim 77, wherein: said at least oneproduct is adipic acid and is biosynthesized from 2-aminopimelate usingat least one polypeptide having an activity chosen fromα-aminotransferase activity, 2-oxoacid decarboxylase activity, synthaseactivity, dehydrogenase complex activity, thioesterase activity,CoA-transferase activity, CoA-ligase activity, and aldehydedehydrogenase activity; said at least one product is adipatesemialdehyde and is biosynthesized from 2-aminopimelate using at leastone polypeptide having an activity chosen from α-aminotransferaseactivity, 2-oxoacid decarboxylase activity, and synthase activity; saidat least one product is 6-aminohexanoic acid and is biosynthesized from2-aminopimelate using a polypeptide having α-aminoacid decarboxylaseactivity; or said at least one product is 6-hydroxyhexanoic acid and isbiosynthesized from 2-aminopimelate using at least one polypeptidehaving an activity chosen from α-aminotransferase activity, 2-oxoaciddecarboxylase activity, synthase activity, and alcohol dehydrogenaseactivity.
 85. The method of claim 84, wherein: said polypeptide having2-oxoacid decarboxylase activity has at least 70% sequence identity tothe amino acid sequence set forth in SEQ ID NO: 34; said polypeptidehaving a-amino acid decarboxylase activity has at least 70% sequenceidentity to the amino acid sequence set forth in any one of SEQ ID NOs:29-34; and/or said polypeptide having thioesterase activity has at least70% sequence identity to the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:
 2. 86. The method of claim 84, wherein said at least oneproduct is adipate semialdehyde and is biosynthesized from2-aminopimelate using at least one polypeptide having an activity chosenfrom a-aminotransferase activity, 2-oxoacid decarboxylase activity, andsynthase activity, and further wherein said method comprises:biosynthesizing 6-aminohexanoic acid from adipate semialdehyde using apolypeptide having ω-transaminase activity; biosynthesizing6-aminohexanoic acid from adipate semialdehyde using a polypeptidehaving ω-transaminase activity and further biosynthesizinghexamethylenediamine from 6-aminohexanoic acid using at least onepolypeptide having an activity chosen from carboxylate reductaseactivity, N-acetyltransferase activity, ω-transaminase activity, anddeacylase activity; biosynthesizing 6-aminohexanoic acid from adipatesemialdehyde using a polypeptide having ω-transminase activity andfurther biosynthesizing caprolactam from 6-aminohexanoic acid using apolypeptide having the activity of a hydrolase; or biosynthesizinghexamethylenediamine from adipate semialdehyde using at least onepolypeptide having carboxylate reductase activity or ω-transaminaseactivity.
 87. The method of claim 86, wherein: said polypeptide havingcarboxylate reductase activity has at least 70% sequence identity to theamino acid sequence set forth in any one of SEQ ID NOs: 3-7; and/or saidpolypeptide having ω-transaminase activity has at least 70% sequenceidentity to the amino acid sequence set forth in any one of SEQ ID NOs:8-13.
 88. The method of claim 84, wherein said at least one product is6-hydroxyhexanoic acid and is biosynthesized from 2-aminopimelate usingat least one polypeptide having an activity chosen fromα-aminotransferase activity, 2-oxoacid decarboxylase activity, synthaseactivity, and alcohol dehydrogenase activity, and further wherein saidmethod comprises: biosynthesizing hexamethylenediamine from6-hydroxyhexanoic acid using at least one polypeptide having an activitychosen from carboxylate reductase activity, ω-transaminase activity, andalcohol dehydrogenase activity; and/or biosynthesizing 1,6-hexanediolfrom 6-hydroxyhexanoic acid using a polypeptide having carboxylatereductase activity and a polypeptide having alcohol dehydrogenaseactivity.
 89. The method of claim 77, wherein said recombinant host is:subjected to a cultivation strategy under aerobic or micro-aerobiccultivation conditions; cultured under conditions of nitrogen,phosphate, or oxygen limitation; and/or retained using a ceramicmembrane to maintain a high cell density during said fermentation. 90.The method of claim 77, further comprising feeding a principal feedstockto the fermentation derived from a biological feedstock or anon-biological feedstock.
 91. The method of claim 90, wherein: saidbiological feedstock is, or derives from monosaccharides, disaccharides,lignocellulose, hemicellulose, cellulose, lignin, levulinic acid andformic acid, triglycerides, glycerol, fatty acids, agricultural waste,condensed distillers' solubles, or municipal waste; or saidnon-biological feedstock is, or derives from, natural gas, syngas,CO₂/H₂, methanol, ethanol, benzoate, non-volatile residue (NVR) or acaustic wash waste stream from cyclohexane oxidation processes, orterephthalic acid I isophthalic acid mixture waste streams.
 92. Themethod of claim 77, wherein said recombinant host is: a prokaryotechosen from the genera Escherichia; Clostridia; Corynebacteria;Cupriavidus; Pseudomonas; Delftia; Bacillus; Lactobacillus; Lactococcus;and Rhodococcus; or a eukaryote chosen from the genera Aspergillus,Saccharomyces, Pichia, Yarrowia, Issatchenkia, Debaryomyces, Arxula, andKluyveromyces.
 93. The method of claim 77, wherein said recombinant hostis: a prokaryote chosen from Escherichia coli, Clostridium ljungdahlii,Clostridium autoethanogenum, Clostridium kluyveri, Corynebacteriumglutamicum, Cupriavidus necator, Cupriavidus metallidurans,Pseudomonasfluorescens, Pseudomonas putida, Pseudomonas oleavorans,Delftia acidovorans, Bacillus subtillis, Lactobacillus delbrueckii,Lactococcus lactis, and Rhodococcus equi; or a eukaryote chosen fromAspergillus niger, Saccharomyces cerevisiae, Pichia pastoris, Yarrowialipolytica, Issathenkia orientalis, Debaryomyces hansenii, Arxulaadenoinivorans, and Kluyveromyces lactis.
 94. The method of claim 77,wherein: said recombinant host exhibits tolerance to high concentrationsof a C6 building block, and wherein the tolerance to high concentrationsof a C6 building block is improved through continuous cultivation in aselective environment; and/or said recombinant host comprises at leastone modification chosen from: increased intracellular concentration ofoxaloacetate for biosynthesis of a C6 building block, wherein saidintracellular concentration is increased in the host by overexpressingrecombinant genes forming oxaloacetate; an imbalance in NADPH that canbe balanced via the formation of a C6 building block; an exogenouslysine biosynthesis pathway synthesizing lysine from 2-oxoglutarate via2-oxoadipate; an exogenous lysine biosynthesis pathway synthesizinglysine from oxaloacetate to meso 2,6-diaminopimelate; attenuatedendogenous degradation pathways of central metabolites and centralprecursors leading to and including C6 building blocks; and the effluxof a C6 building block across the cell membrane to the extracellularmedia is enhanced or amplified by genetically engineering structuralmodifications to the cell membrane or increasing any associatedtransporter activity for a C6 building block.
 95. A compositioncomprising: at least one bioderived 6-carbon compound chosen from adipicacid, adipate semialdehyde, 6-aminohexanoic acid, 6-hydroxyhexanoicacid, caprolactam, hexamethylenediamine, and 1,6-hexanediol, whereinsaid at least one bioderived 6-carbon compound is biosynthesized usingthe method of claim 77; and a compound other than the bioderived6-carbon compound.
 96. A biobased polymer or resin comprising at leastone bioderived 6-carbon compound chosen from adipic acid, adipatesemialdehyde, 6-aminohexanoic acid, 6-hydroxyhexanoic acid, caprolactam,hexamethylenediamine, and 1,6-hexanediol, wherein said at least onebioderived compound is biosynthesized using the method of claim
 77. 97.A molded product obtainable by molding a biobased polymer or resin ofclaim
 93. 98. A process for producing a biobased polymer or resincomprising biosynthezing at least one bioderived 6-carbon compoundchosen from adipic acid, adipate semialdehyde, 6-aminohexanoic acid,6-hydroxyhexanoic acid, caprolactam, hexamethylenediamine, and1,6-hexanediol using the method of claim 77, wherein said method furthercomprises: culturing or growing a host cell under conditions and for asufficient period of time to produce said at least one bioderived6-carbon compound; and chemically reacting said at least one bioderived6-carbon compound with itself or another compound in a polymer-producingor resin-producing reaction.
 99. A biochemical network comprisingmeso-2,6-diaminopimelate and at least one enzyme chosen from adehydrogenase, a CoA-transferase, a dehydratase, a reductase, a mutase,a CoA-ligase, an ammonia lyase, and a thioesterase, wherein said atleast one enzyme is capable of enzymatically converting themeso-2,6-diaminopimelate to 2-aminopimelate, and further wherein saidbiochemical network optionally comprises: an α-aminotransferase, whereinsaid α-aminotransferase is capable of enzymatically converting2-aminopimelate to 2-oxo-pimelate; an α-aminotransferase and at leastone enzyme chosen from a synthase, a dehydrogenase complex, and adecarboxylase, wherein: said α-aminotransferase is capable ofenzymatically converting 2-aminopimelate to 2-oxo-pimelate; and said atleast one enzyme is capable of enzymatically converting 2-oxo-pimelateto adipyl-CoA or adipate semialdehyde; an α-aminotransferase, at leastone first enzyme chosen from decarboxylase, a synthase, and adehydrogenase complex, and at least one second enzyme chosen from adehydrogenase, a CoA-transferase, a CoA-ligase, and a thioesterase,wherein: said α-aminotransferase is capable of enzymatically converting2-aminopimelate to 2-oxo-pimelate; said at least one first enzyme iscapable of enzymatically converting 2-oxo-pimelate to adipyl-CoA oradipate semialdehyde; and said at least one second enzyme is capable ofenzymatically converting adipyl-CoA or adipate semialdehyde to adipicacid; a decarboxylase, wherein said decarboxylase is capable ofenzymatically converting 2-aminopimelate to 6-aminohexanoic acid; adecarboxylase and at least one enzyme chosen from a hydrolase, areductase, a transaminase, an N-acetyltransferase, and a deacetylase,wherein: said decarboxylase is capable of enzymatically converting2-aminopimelate to 6-aminohexanoic acid; and said at least one enzyme iscapable of enzymatically converting 6-aminohexanoic acid into at leastone of caprolactam or hexamethylenediamine; at least one enzyme chosenfrom an aminotransferase, a synthase, a decarboxylase, and adehydrogenase, wherein said at least one enzyme is capable ofenzymatically converting 2-aminopimelate to 6-hydroxyhexanoic acid; orat least one first enzyme chosen from an aminotransferase, a synthase, adecarboxylase, and a dehydrogenase and at least one second enzyme chosenfrom a reductase, a transaminase, or an alcohol dehydrogenase, wherein:said at least one first enzyme is capable of enzymatically converting2-aminopimelate to 6-hydroxyhexanoic acid; and said at least one secondenzyme is capable of enzymatically converting 6-hydroxyhexanoic acidinto at least one of hexamethylenediamine and 1,6-hexanediol.